Biomaterial comprising hyaluronic acid and derivatives thereof in interpenetrating polymer networks (IPN)

ABSTRACT

Provided is a biomaterial comprising an interpenetrating polymer network (IPN), wherein one of the polymer components is an acidic polysaccharide or a semi-synthetic derivative thereof. The polysaccharide can be hyaluronic acid, and the second polymer component can be a non-toxic, non-carcinogenic synthetic chemical polymer. The derivative can be a total or partial hyaluronic acid ester or a hyaluronic acid salt. The ester or salt can be formed with a pharmacologically active molecule. Methods for preparing the IPN are also disclosed. The acidic polysaccharide or derivative thereof and the synthetic chemical polymer comprising the IPN can be cross-linked, or the synthetic chemical polymer can be grafted onto the acidic polysaccharide. The cross-linking or grafting can be achieved using compounds capable of generating radicals, or via functional groups on the acidic polysaccharide and the synthetic chemical polymer. The IPN can be formed prior to cross-linking or grafting. The IPN biomaterial can be in the form of a film, a membrane, a sponge, a hydrogel, a guide channel, a thread, a gauze, or a non-woven tissue. Such IPN biomaterials can be used in the biomedical and sanitary fields, including dermatology, urology, orthopedics, otologic microsurgery, otoneurology, functional, post-traumatic and rhinosinusal endoscopic microsurgery, plastic surgery, and in the cardiovascular system.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to interpenetrating polymer networkswherein one of the components is an acidic polysaccharide or aderivative thereof, a process for their preparation, and their use asbiomaterials for biomedical and sanitary applications.

2. Description of Related Art

Hyaluronic acid (HA) is a natural heteropolysaccharide composed ofalternate residues of D-glucuronic acid and N-acetyl-D-glucosamine. Itis a linear polymer with a molecular weight of between 50,000 and13,000,000, depending upon the source from which it is obtained and howit is prepared and analyzed. In nature, it is present in pericellulargels, in the fundamental substance of connective tissues in vertebrateorganisms, of which it is one of the main components, in the synovialfluid of joints, the vitreous humor, umbilical cord tissues, and incocks'to combs.

Specific fractions of hyaluronic acid with definite molecular weightsare known which do not possess inflammatory activity, and which cantherefore be used to facilitate wound healing or to substitute theendobulbar fluids or in therapy for joint pathologies by intra-articularinjection, as described in European Patent No. 0 138 572 granted to thepresent Applicants.

Also known are esters of hyaluronic acid, in which all or part of thecarboxy groups of the acid are esterified, and their use inpharmaceuticals and cosmetics, and in biodegradable plastic materials,as described in U.S. Pat. Nos. 4,851,521 and 4,965,353, also granted tothe present Applicants.

It is a known fact that the application of hyaluronic acid is able toaccelerate healing in patients with bedsores, wounds and burns. Its rolein the various stages of wound healing has been described, with the aidof a theoretical model, by Weigel et al. ("A model for the role ofhyaluronic acid and fibrin in the early events during the inflammatoryresponse and wound healing," J. Theor. Biol., 119: 219, 1986).

Studies aimed at obtaining products (biomaterials) for medical, sanitaryand pharmaceutical applications, composed of hyaluronic acid esters oresters of other polysaccharides used as such or in mixtures with otherpolymers, have led to the creation of various products. These includetissues such as gauzes of varying density (number of threads percentimeter), of varying dimensions and denier (weight per 9,000 metersof thread), films, membranes, gels, guide channels, etc. Some examplesof films appear in two patents granted to the present Applicants, namelyU.S. Pat. Nos. 4,851,521 and 4,965,353. The use of such materials islimited by the impossibility of using moulding systems for theirconstruction and use.

An interpenetrating polymer network,. IPN, is an intimate combination oftwo polymers, both in network form, at least one of which is synthesizedor cross-linked in the immediate presence of the other. If one of thetwo polymers is in network form (crosslinked) and the other is a linearpolymer (not crosslinked), a semi-IPN results (L. H. Sperling,Interpenetrating Polymer Networks, CHEMTECH, February, 1988. The termIPN currently covers new materials where the two polymers in the mixtureare not necessarily bound together, but the components are physicallyassociated. Clearly, these new materials open up the possibility ofgiving physical, mechanical and manufacturable properties to easilydegradable polymers, creating new materials wherein new biologicalproperties can be coupled with their mechanical properties. Examples ofnewly-developed IPN and applications thereof have been reported whereinone of the two components is a water-soluble polymer (U.S. Pat. Nos.4,678,468 and 4,747,953).

IPN comprising a naturally occurring polymer and a synthetic polymer foruse in the medical, sanitary, and pharmaceutical areas is, however,novel to the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a biomaterial,comprising an interpenetrating polymer network, IPN, wherein one of thecomponents is an acidic polysaccharide or a derivative thereof. Saidacidic polysaccharide can be hyaluronic acid, and the second polymercomponent can be a non-toxic, non-carcinogenic synthetic chemicalpolymer. Said derivative can be a total or partial hyaluronic acid esteror hyaluronic acid salt. Said ester or salt can be formed with apharmacologically active molecule. Also disclosed are methods forpreparing the IPN of the present invention.

Another object of the present invention is to provide IPN wherein saidacidic polysaccharide and said synthetic chemical polymer comprisingsaid IPN are crosslinked, or wherein said synthetic chemical polymer isgrafted onto said acidic polysaccharide. The crosslinking or graftingcan be achieved using compounds capable of generating radicals, or viafunctional groups on said acidic polysaccharide and said syntheticchemical polymer. Said IPN can be formed prior to crosslinking orgrafting, and possess strong interactions between the natural andsynthetic polymers therein by virtue of the grafting of the syntheticpolymer onto the natural one or the intermolecular crosslinks betweenthe two.

Yet another object of the present invention is to provide said IPN in aform selected from the group consisting of a film, a membrane, a sponge,a hydrogel, a guide channel, a thread, a gauze, and a non-woven tissue.

A further object of the present invention is the use of said IPN in thebiomedical and sanitary fields, including their use in dermatology,urology, orthopedics, otologic microsurgery, otoneurology, functional,post-traumatic and rhinosinusal endoscopic microsurgery, plasticsurgery, in the cardiovascular system, and in any other type of surgeryin which their properties are useful.

The IPN of the present invention possess particular, improved chemicaland physical characteristics as compared to conventional IPN. Thesebiomaterials retain the biocompatible characteristics of thepolysaccharide, hyaluronic acid, or ester derivative thereof used as oneof the two components of the IPN, as well as the mechanicalcharacteristics of the chemical polymer used as the second of the twopolymeric components of the IPN.

With respect to the hyaluronic acid esters useful in the presentinvention, these esters can be used alone or in association with otheractive principles such as therapeutic agents like antiinfective agents,antibiotics, antimicrobials, antiinflammatory agents, cytostatic agents,cytotoxic agents, antiviral agents, anaesthetic agents, antiseptics, anddisinfectants.

With respect to the chemical polymers useful in the present invention,any of those already employed as biomaterials can be used to produce theIPN disclosed infra. Such chemical polymers are described in G. W.Hastings, Macromolecular Biomaterials, CRC Press, 1984, and S. D. Bruck,Properties of Biomaterials in the Physiological Environment, CRC Press,1980. The only factor limiting the use of such synthetic polymers isthat they must be non-toxic and non-carcinogenic.

For purely illustrative purposes, presented below are some examplesdescribing the preparation of IPN according to the present invention.

Further scope of the applicability of the present invention will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention, as modifications and variations in theembodiments herein discussed may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

The contents of each of the references cited herein are incorporated byreference in their entirety.

Preparation of Hyaluronic Acid Fractions Useful in the IPN of thePresent Invention

EXAMPLE 1

Method of preparing a mixture of hyalastine and hyalectin having noinflammatory activity

Fresh or frozen cocks' combs, (3000 g) are minced in a meat mincer andthen carefully homogenized in a mechanical homogenizer. The paste thusobtained is placed in a stainless steel container AISI 316 or in glassand treated with 10 volumes of anhydrous acetone. The whole is agitatedfor 6 hours at a speed of 50 rpm. It is left to separate for 12 hoursand the acetone is discarded by syphoning. The acetone extraction isrepeated until the discarded acetone has reached the correct degree ofhumidity (Karl-Fischer method). The whole is then centrifuged and vacuumdried at a suitable temperature for 5-8 hours. In this way about 500-600g of dry powdered cocks' combs are obtained.

300 g of dry powder are exposed to enzymatic digestion with papain (0.2g) under aqueous conditions, buffered with phosphate buffer in thepresence of a suitable quantity of cysteine hydrochloride. The resultantis agitated for 24 hours at 60 rpm keeping the temperature constant at60°-65° C. It is then cooled to 25° C. and Celite® (60 g) is added,maintaining the agitation for another hour. The resulting mixture isfiltered until a clear liquid is obtained. The clear liquid thenundergoes molecular ultrafiltration using membranes with a molecularexclusion limit of 30,000 in order to retain on the membrane thosemolecules with a molecular weight greater than 30,000.

The product is ultrafiltered from 5 to 6 original volumes addingdistilled water continually to the product in ultrafiltration. Theaddition of water is suspended and the ultrafiltration is continueduntil the volume is reduced to 1/3 of the original volume.

The residual liquid is rendered 0.1M by the addition of sodium chlorideand the temperature is brought to 50° C. Under agitation at 60 rpm, 45 gof cetylpyridinium chloride are added. It is agitated for 60 minutes andthen 50 g of Celite® are added. Under agitation, the temperature of thewhole is brought to 25° C. and the precipitate formed by centrifugationis gathered. The precipitate obtained is suspended in a 0.01M solutionof sodium chloride (5 liters) containing 0.05% of cetylpyridiniumchloride. The resulting suspension is agitated for 60 minutes at 50° C.;the temperature is then brought to 25° C. and the precipitate iscentrifuged. The washing operation is repeated 3 times after which theprecipitate is gathered in a receptacle containing 3 liters of a 0.05Msolution of sodium chloride containing 0.05% of cetylpyridiniumchloride. It is agitated at 60 rpm for 60 minutes and the temperature iskept constant at 25° C. for two hours. The supernatant is eliminated bycentrifugation. The procedure is repeated several times with solutionsof 0.1M sodium chloride containing 0.05% of cetylpyridinium chloride.The mixture is centrifuged and the supernatant is discarded. Theprecipitate is dispersed in a solution of 0.30M sodium chloridecontaining 0.05% of cetylpyridinium chloride (3 liters). The mixture isagitated and both the precipitate and the clear liquid are gathered.Extraction is repeated three more times on the precipitate, each timeusing 0.5 liter of the same aqueous solution.

Finally, the precipitate residue is eliminated and the clear liquids areall placed together in a single container. The temperature of the liquidis brought to 50° C. under constant agitation. The liquid is thenbrought to 0.23M with sodium chloride. 1 g of cetylpyridinium chlorideis added and it is maintained in agitation for 12 hours.

The mixture is cooled at 25° C. and then filtered first on Celite® packand then through a filter. It then undergoes molecular ultrafiltrationagain, on a membrane with a molecular exclusion limit of 30,000,ultrafiltering three initial volumes with the addition of a solution of0.33M sodium chloride. The addition of sodium chloride solution isinterrupted and the volume is reduced to 1/4 of the initial volume. Thesolution thus concentrated is precipitated under agitation (60 rpm) at25° C. with 3 volumes of ethanol (95%). The precipitate is gathered bycentrifugation and the supernatant is discarded. The precipitate isdissolved in 1 liter of 0.01M sodium chloride and the precipitation isrepeated with 3 volumes of 95% ethanol.

The precipitate is gathered and washed first with 75% ethanol (3 times),then with absolute ethanol (3 times), and lastly with absolute acetone(3 times).

The product thus obtained (HYALASTINE+HYALECTIN fractions) has anaverage molecular weight of between 250,000 and 350,000.

The yield of HA is 0.6% of the original fresh tissue.

EXAMPLE 2

Method of preparing the hyalastine fraction from the mixture obtained bythe method described in Example 1

The mixture obtained by the method described in Example 1 is dissolvedin twice distilled apyrogenic water at the rate of 10 mg of product toeach 1 ml of water. The solution obtained is exposed to molecularfiltration through filter membranes with a molecular exclusion limit of200,000, following a concentration technique on the membrane without theaddition of water. During the ultrafiltration process through membraneswith a molecular exclusion limit of 200,000, the molecules with amolecular weight of more than 200,000 do not pass through, while thesmaller molecules pass through the membrane together with the water.During the filtration procedure no water is added, so that the volumedecreases, and there is therefore an increase in the concentration ofmolecules with a molecular weight of more than 200,000. The product isultrafiltered until the volume on top of the membrane is reduced to 10%of the initial volume. Two volumes of apyrogenic twice distilled waterare added and it is then ultrafiltered again until the volume is reducedto 1/3. The operation is repeated twice more. The solution passedthrough the membrane is brought to 0.1M with sodium chloride and thenprecipitated with 4 volumes of 95% ethanol. The precipitate is washed 3times with 75% ethanol and then vacuum dried.

The product thus obtained (HYALASTINE fraction) has an average molecularweight of between 50,000 and 100,000. The yield of HA is equal to 0.4%of the original fresh tissue.

Hyaluronic acid with an average molecular weight of 155,000 can besimilarly obtained by employing appropriate ultrafiltration membranesand gel filtration chromatography.

EXAMPLE 3

Method of obtaining the hyalectin fraction

The concentrated solution gathered in the container on top of theultrafiltration membrane with a molecular exclusion of 200,000 as inExample 2, is diluted with water until a solution containing 5 mg/ml ofhyaluronic acid is obtained, as determined by quantitative analysisbased on the amount of glucuronic acid.

The solution is brought to 0.1M in sodium chloride and then precipitatedwith 4 volumes of 95% ethanol. The precipitate is washed 3 times with75% ethanol and then vacuum dried.

The product thus obtained (HYALECTIN fraction) has an average molecularweight of between 500,000 and 730,000. This corresponds to a specificfraction of hyaluronic acid with a defined molecular chain length ofabout 2,500 to 3,500 saccharide units with a high degree of purity. Theyield of HA is equal to 0.2% of the original fresh tissue.

EXAMPLE 4

Preparation of the tetrabutylammonium salt of hyaluronic acid

4.02 g of HA sodium salt (10 m.Eq.) are solubilized in 400 ml ofdistilled H₂ O. The solution is then eluted in a thermostatic column at4° C. containing 15 ml of sulphonic resin (Dowex 50×8) intetrabutylammonium form. The eluate, free from sodium, is instantlyfrozen and freeze-dried. Yield: 6.18 g.

EXAMPLE 5

Preparation of a film of hyaluronic acid (HA) and polyacrylic acid

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water byshaking at a temperature of 50° C. for 30 minutes. A 1% solution of HAis thus obtained, which will be referred to as solution A.

80 mg of PAA (MW 250,000) are dissolved in 8 ml of distilled water byshaking at a temperature of 50° C. for 12 hours. A 1% solution of PAA isthus obtained, which will be referred to as solution B1, and this isleft to cool to room temperature.

Solution A is slowly added to solution B1 while being continuouslyshaken at room temperature. Shaking is continued for one hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PAA in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenset at 75° C. Once the solvent has evaporated, a transparent andhomogeneous film is obtained.

EXAMPLE 6

Preparation of a film of hyaluronic acid (HA) and polyvinylpyrrolidone(PVP)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water byshaking at a temperature of 50° C. for 30 minutes. A 1% solution of HAis obtained, which is referred to as solution A.

80 mg of PVP (MW 40,000) are dissolved in 8 ml of distilled water, byshaking at a temperature of 50° C. for 5 hours. A 1% solution of PVP isobtained, referred to as solution B2, which is left to cool to roomtemperature.

Solution A is slowly added to solution B2 while shaking at roomtemperature. The solution is shaken for one hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PVP in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed into a ventilationoven set at a temperature of 75° C. Once the solvent has completelyevaporated, a transparent and homogeneous film is obtained.

EXAMPLE 7

Preparation of a film of hyaluronic acid (HA) and polyacrylamide (PAAm)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water, whileshaking at a temperature of 50° C. for 30 minutes. A 1% solution of HAis obtained, which will be referred to as solution A.

80 mg of PAAm (MW 5×10⁶) are dissolved in 8 ml of distilled water, whileshaking at a temperature of 50° C. for 12 hours. A 1% solution of PAAmis thus obtained, referred to as solution B3, which is then left to coolto room temperature.

Solution A is slowly added to solution B3 while being continuouslyshaken at room temperature. The solution is shaken for 1 hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PAAm in a weight ratio of20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at 75° C. Once the solvent has completelyevaporated, a transparent and homogeneous film is obtained.

EXAMPLE 8

Preparation of a film of hyaluronic acid (HA) and polyethylene oxide(PEO)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water, whileshaking at a temperature of 50° C. for 30 minutes. A 1% solution of HAis thus obtained, which will be referred to as solution A.

80 mg of PEO (MW 100,000) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 12 hours. A 1% solution ofPEO is thus obtained, referred to as solution B4, which is then left tocool to room temperature.

Solution A is slowly poured into solution B4 while being continuouslyshaken at room temperature. The solution is shaken for one hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PEO in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenat a temperature of 75° C. Once the solvent has completely evaporated, atransparent and homogeneous film is obtained.

EXAMPLE 9

Preparation of a film of hyaluronic acid (HA) and Mowiol (vinylalcohol-vinyl acetate copolymer, MOW)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is obtained, which will be referred to as solution A.

80 mg of MOW (MW 127,000) are dissolved in 8 ml of distilled water,while being shaken at a temperature of 50° C. for 12 hours. A 1%solution of MOW is thus obtained, referred to as solution B5, which isthen left to cool to room temperature.

Solution A is slowly added to solution B5 while being continuouslyshaken at room temperature. The solution is shaken for 1 hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/MOW in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenset at a temperature of 75° C. Once the solvent has completelyevaporated, a transparent and homogeneous film is obtained.

EXAMPLE 10

Preparation of a film of hyaluronic acid (HA) and polyvinyl alcohol(PVA)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water, whileshaking at a temperature of 50° C. for 30 minutes. A 1% solution of HAis thus obtained, which will be referred to as solution A.

80 mg of PVA (MW 115,000) are placed in a flask, with a reflux coolingsystem and a magnetic stirrer, containing 8 ml of distilled water. Theflask is placed in an oil bath at a temperature of 150° C. and shakenfor 5 hours. Once the PVA has completely dissolved, a 1% solution of PVAis obtained, which will be referred to as solution B6; this is left tocool to room temperature.

Solution A is slowly added to solution B6 under constant shaking at roomtemperature. The solution is shaken for 1 hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PVA in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenset at a temperature of 75° C. Once the solvent has completelydissolved, a transparent and homogeneous film is obtained.

EXAMPLE 11

Preparation of a film of hyaluronic acid (HA) and polyphosphazenes, forexample poly-(trifluoroethoxy)phosphazene (PF1)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is thus obtained, which will be referred to as solution A. 80 mg ofPF1 (MW 15,000) are dissolved in 8 ml of distilled water while beingshaken at a temperature of 50° C. for 12 hours. A 1% solution of PFI isthus obtained, which will be referred to as solution B7; this is left tocool to room temperature.

Solution A is slowly added to solution B7 while being constantly shakenat room temperature. The solution is shaken for one hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PFI in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenset at a temperature of 75° C. Once the solvent has completelyevaporated, a transparent and homogeneous film is obtained.

EXAMPLE 12

Preparation of a film of hyaluronic acid (HA) and polyphosphazenes, forexample poly-(di(p-sodio sulfoxy phenoxy)phosphazene (PF2)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of obtained, which will be referred to assolution A.

80 mg of PF2 (MW 15,000) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 5 hours. A 1% solution ofPF2 is thus obtained, which will be referred to as solution B8; this isleft to cool to room temperature.

Solution A is slowly added to solution B8 while being continuouslyshaken at room temperature. The solution is shaken for one hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PF2 in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a ventilation ovenset at a temperature of 75° C. Once the solvent has completelyevaporated, a transparent and homogeneous film is obtained.

The following examples illustrate the preparation of sponges by thefreeze-drying of mixtures of hyaluronic acid and water-soluble polymers.

EXAMPLE 13

Preparation of a freeze-dried sponge containing hyaluronic acid (HA) andpolyacrylic acid (PAA)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is thus obtained, which will be referred to as solution A, and whichis left to cool to room temperature.

80 mg of PAA (MW 250,000) are dissolved in 8 ml of distilled water,while being shaken at a temperature of 50° C. for 12 hours. A 1%solution of PASA is thus obtained, referred to as solution B1, which isleft to cool to room temperature.

Solution A is slowly added to solution B1 while shaking at roomtemperature. The solution is shaken for 1 hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PAA in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a freeze-drier.Lyophilization is achieved by freezing to -30° C. at atmosphericpressure, then heating to -20° C. in a vacuum (0.015 mbar) for 24 hours.Once the freeze-drying process is complete, a soft, white, spongymaterial is obtained.

EXAMPLE 14

Preparation of a freeze-dried sponge containing hyaluronic acid (HA) andpolyvinylpyrrolidone (PVP)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water at atemperature of 50° C. for 30 minutes. A 1% solution of HA is obtained,which will be referred to as solution A; this is left to cool no roomtemperature.

80 mg of PVP (MW 40,000) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 12 hours. A 1% solution ofPVP is thus obtained which will be referred to as solution B2; this isleft to cool to room temperature.

Solution A is slowly added to solution B2 while being constantly shakenat room temperature. The solution is shaken for 1 hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PVP in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a freeze-drier.Lyophilization is achieved by freezing to -30° C. at atmosphericpressure, then heating to -20° C. in a vacuum (0.015 mbar) for 24 hours.Once the freeze-drying process is complete, a soft, white, spongymaterial is obtained.

EXAMPLE 15

Preparation of a freeze-dried sponge containing hyaluronic acid (HA) andpolyacrylamide (PAAm)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is thus obtained, which will be referred to as solution A; this isleft to cool to room temperature.

80 mg of PAAm (MW 5×10⁶) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 12 hours. A 1% solution ofPAAm is thus obtained, which will be referred to as solution B3; this isleft to cool to room temperature.

Solution A is slowly added to solution B3 while being continually shakenat room temperature. The solution is shaken for 1 hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PAAm in a weight ratio of20/80 is poured into a polystyrene Petri dish and placed in afreeze-drier. Lyophilization is achieved by freezing to -30° C. atatmospheric pressure, and then heating to -20° C. in a vacuum (0.015mbar) for 24 hours. Once the freeze-drying process is complete a soft,white, spongy material is obtained.

EXAMPLE 16

Preparation of a freeze-dried sponge containing hyaluronic acid (HA) andpolyethylene oxide (PEO)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is thus obtained, which will be referred to as solution A; this isleft to cool to room temperature.

80 mg of PEO (MW 100,000) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 12 hours. A solutionreferred to as B4 is obtained; this is left to cool to room temperature.

Solution A is slowly added to solution B4 while being continuouslyshaken at room temperature. The solution is shaken for 1 hour to allowcomplete amalgamation of the two components.

The solution containing the mixture of HA/PEO in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a freeze-drier.Lyophilization is achieved by freezing to -30° C. at atmosphericpressure, and then heating to -20° C. in a vacuum (0.015 mbar) for 24hours. Once the freeze-drying process is complete, a soft, white, spongymaterial is obtained.

EXAMPLE 17

Preparation of a freeze-dried sponge containing hyaluronic acid (HA) andpolyphosphazenes, for example poly(methoxy ethoxy)phosphazene (PF3)

20 mg of HA (MW 155,000) are dissolved in 2 ml of distilled water whilebeing shaken at a temperature of 50° C. for 30 minutes. A 1% solution ofHA is thus obtained, which will be referred to as solution A; this isleft to cool to room temperature.

80 mg of PF3 (MW 15,000) are dissolved in 8 ml of distilled water whilebeing shaken at a temperature of 50° C. for 12 hours. A 1% solution ofPF3 is thus obtained, which will be referred to as solution B5; this isleft to cool to room temperature.

Solution A is slowly added to solution B5 while being shaken at roomtemperature. The solution is shaken for one hour to allow completeamalgamation of the two components.

The solution containing the mixture of HA/PF3 in a weight ratio of 20/80is poured into a polystyrene Petri dish and placed in a freeze-drier.Lyophilization is achieved by freezing to -30° C. at atmosphericpressure, followed by heating to -20° C. in a vacuum for 24 hours. Oncethe freeze-drying process is complete, a soft, white, spongy material isobtained.

EXAMPLE 18

Preparation of a hydrogel containing hyaluronic acid (HA) and polyvinylalcohol (PVA) 20 mg of HA (MW 155,000) are dissolved in 2 ml ofdistilled water while being shaken at a temperature of 50° C. for 30minutes. A 1% solution of HA is obtained, which will be referred to assolution A.

320 mg of PVA (MW 115,000) are placed in a flask, with a reflux coolingsystem and a magnetic stirrer, containing 8 ml of distilled water. Theflask is placed in an oil bath at a temperature of 150° C. for 5 hourswhile being shaken. Once the PVA has completely dissolved, a 4% solutionof PVA is obtained, which will be referred to as solution B6; this isleft to cool to room temperature.

Solution A is slowly added to 2 ml of solution B6 while being constantlyshaken at room temperature. The solution containing the mixture ofHA/PVA in a weight ratio of 20/80 is shaken for one hour to allowcomplete amalgamation of the two components.

The solution of HA/PVA is poured into a polystyrene Petri dish andexposed to 5 freeze-thaw cycles. Each cycle consists of bringing thesample from room temperature to -20° C., keeping the sample at thistemperature for one hour, thawing it by bringing it back to roomtemperature, and completing the cycle by keeping it at room temperaturefor one hour.

By the second cycle, the solution already has a gelatinous appearance,and its consistency increases with subsequent cycles. At the end of thefifth cycle, the sample has a uniform, white, rubbery consistency, andits water content is about 60%.

The following examples illustrate the preparation of films by theevaporation of the solvent from mixtures of hyaluronic acid (HA)dissolved in dimethyl sulfoxide (DMSO) and polymers soluble in DMSO.

EXAMPLE 19

Preparation of a film containing hyaluronic acid (HA1) and polyvinylalcohol (PVA) HA is dissolved in DMSO as follows: 100 mg of HA (MW155,000) are dissolved in distilled water while being shaken at roomtemperature for 30 minutes. The water is then substituted with DMSO byadding the second solvent to the solution and heating to 90° C. toevaporate the water. Further DMSO is added to bring the solution to afinal volume of 10 ml. A 1% solution of HA in DMSO is thus obtained,which will be referred to as solution A in Examples 15-17.

100 mg of PVA are dissolved in DMSO while being shaken at a temperatureof 100° C. for 1 hour and then brought to a final volume of 10 ml. A 1%solution of PVA is thus obtained, which will be referred to as solutionB. Solution A is slowly added to solution B while being continuouslyshaken at a temperature of 100° C. The resulting solution is shaken forone hour to allow complete amalgamation of the two components.

The resulting solution containing the mixture of HA/PVA in a weightratio of 20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at a temperature of 75° C. Once the solvent hascompletely evaporated, a transparent, homogeneous film is obtained.

EXAMPLE 20

Preparation of a film containing hyaluronic acid (HA) and Clarene L6,Solvay (C1 L6), ethylene-vinyl alcohol copolymer with a low ethylenecontent (29 mole %) 5 100 mg of C1 L6 are dissolved in DMSO whileshaking for 1 hour and brought to a final volume of 10 ml. A 1% solutionof C1 L6 is obtained, which will be referred to as solution C.

Solution A is slowly added to solution C while being continuously shakenat a temperature of 70° C. The resulting solution is shaken for 1 hourto allow complete amalgamation of the two components.

The resulting solution containing the mixture of HA/C1 L6 in a weightratio of 20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at a temperature of 75° C. Once the solvent hascompletely evaporated, a transparent and homogeneous film is obtained.

EXAMPLE 21

Preparation of a film containing hyaluronic acid (HA) and Clarene P10,Solvay (C1 P10), ethylene-vinyl alcohol copolymer with a medium ethylenecontent (36 mole %)

100 mg of C1 P10 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and then brought to a final volume of10 ml. A 1% solution of C1 P10 is thus obtained, which will be referredto as solution D.

Solution A is slowly added to solution D while being continuously shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution containing the mixture of HA/C1 P10 in a weightratio of 20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at a temperature of 75° C. Once the solvent hascompletely evaporated, a transparent and homogeneous film is obtained.

EXAMPLE 22

Preparation of a film containing hyaluronic acid (HA) and Clarene R20,Solvay (C1 R20), ethylene-vinyl alcohol copolymer with a high ethylenecontent (40 mole %)

100 mg of C1 R20 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and brought to a final volume of 10ml. A 1% solution of C1 R20 is thus obtained, which will be referred toas solution E.

Solution A is slowly added to solution E under continuous shaking at atemperature of 70° C. The resulting solution is shaken for one hour toallow complete amalgamation of the two components.

The solution containing the mixture of HA/C1 R20 in a weight ratio of20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at a temperature of 75° C. Once the solvent hascompletely evaporated, a transparent and homogeneous film is obtained.

EXAMPLE 23

Preparation of a film containing hyaluronic acid (HA) and polyurethane(PU), Cardiomat 610, Contron

0.670 ml, of a 15% solution of PU in tetrahydrofuran:dioxan, 1:1, aredissolved in 8 ml of DMSO while being shaken at a temperature of 70° C.for one hour. When the starting solvents have evaporated, the solutionis brought to a final volume of 10 ml with DMSO. A 1% solution of PU isthus obtained, which will be referred to as solution F.

Solution A is slowly added to solution F while being continuouslystirred at a temperature of 70° C. The resulting solution is shaken forone hour to allow complete amalgamation of the two components.

The resulting solution containing HA/PU in a weight ratio of 20/80 ispoured into a polystyrene Petri dish and placed into a ventilation ovenset at 75° C. Once the solvent has completely evaporated, a transparentand homogeneous film is obtained.

EXAMPLE 24

Preparation of a film containing hyaluronic acid (HA) and polylacticacid (PLA)

100 mg of PLA are dissolved in DMSO while being shaken at a temperatureof 85° C. for one hour and then brought to a final volume of 10 ml. A 1%solution of PLA is thus obtained, which will be referred to as solutionG.

Solution A is slowly added to solution G while being continuously shakenat a temperature of 85° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HA/PLA in a weightratio of 20/80 is poured into a polystyrene Petri dish and placed in aventilation oven set at a temperature of 75° C. Once the solvent hascompletely evaporated, a transparent and homogeneous film is obtained.

Use of Hyaluronic Ester Derivatives in IPN

The present IPN can be prepared using not only hyaluronic acid, butester derivatives of hyaluronic acid as well. The preparation of suchesters is described in U.S. Pat. No. 4,851,521.

Ester derivatives of hyaluronic acid useful in the present invention areesters of hyaluronic acid with aliphatic, araliphatic, cycloaliphatic orheterocyclic alcohols, in which are esterified all (so-called "totalesters") or only a part (so-called "partial esters") of the carboxylicgroups of the hyaluronic acid, and salts of the partial esters withmetals or with organic bases, biocompatible or acceptable from apharmacological point of view.

The useful esters include esters which derive from alcohols whichthemselves possess a notable pharmacological action. The saturatedalcohols of the aliphatic series or simple alcohols of thecycloaliphatic series are useful in the present invention.

In the above-mentioned esters in which some of the carboxylic acidgroups remain free (i.e., partial esters), these may be salified withmetals or organic bases, such as with alkaline or alkaline earth metalsor with ammonia or nitrogenous organic bases.

Alcohols of the aliphatic series to be used as esterifying components ofthe carboxylic groups of hyaluronic acid for use in the IPN according tothe present invention are, for example, those with a maximum of 14carbon atoms, which may be saturated or unsaturated, and which maypossibly also be substituted with other free functional or functionallymodified groups, such as amine, hydroxyl, aldehyde, ketone, mercaptan,or carboxyl groups, or by groups derived from these, such as hydrocarbylor di-hydrocarbylamine groups (the term "hydrocarbyl" refers not only tomonovalent radicals of hydrocarbons such as the C_(n) H_(2n+1) type, butalso bivalent or trivalent radicals, such as "alkylenes," C_(n) H_(2n),or "alkylidenes," C_(n) H_(2n), ether or ester groups, acetal or ketalgroups, thioether or thioester groups, and esterified carboxyl orcarbamide groups and carbamide substituted with one or more hydrocarbylgroups, with nitrile groups, or with halogens.

Of the above-mentioned groups containing hydrocarbyl radicals, these arepreferably lower aliphatic radicals, such as alkyls, with a maximum of 6carbon atoms. Such alcohols may also be interrupted in the carbon atomchain by heteroatoms, such as oxygen, nitrogen and sulfur atoms.Preferred are alcohols substituted with one or two of the saidfunctional groups.

Alcohols of the above-mentioned group which are preferably used arethose with a maximum of 14, and especially 6 carbon atoms, and in whichthe hydrocarbyl atoms in the above-mentioned amine, ether, ester,thioether, thioester, acetal, or ketal groups represent alkyl groupswith a maximum of 4 carbon atoms, and also in the esterified carboxyl orsubstituted carbamide groups the hydrocarbyl groups are alkyls with thesame number of carbon atoms, and in which in the amine or carbamidegroups may be alkylenamine or alkylencarbamide groups with a maximum of8 carbon atoms. Of these alcohols, specifically preferred are saturatedand non-substituted alcohols, such as methyl, ethyl, propyl, andisopropyl alcohols, normal butyl alcohol, isobutyl alcohol, tertiarybutyl alcohol, amyl, pentyl, hexyl, octyl, nonyl and dodecyl alcohols,and those with a linear chain, such as normal octyl and dodecylalcohols. Of the substituted alcohols of this group, the bivalentalcohols are ethyleneglycol, propyleneglycol and butyleneglycol, thetrivalent alcohols such as glycerine, the aldehyde alcohols such astartronic alcohol, the carboxylic alcohols such as lactic acids, forexample glycolic acid, malic acid, the tartaric acids, citric acid, theaminoalcohols, such as normal aminoethanol, aminopropanol, normalaminobutanol and their dimethylated and diethylated derivatives in theamine function, choline, pyrrolidinylethanol, piperidinylethanol,piperazineylethanol and the corresponding derivatives of normal propylor normal butyl alcohol, monothioethyleneglycol or its alkylderivatives, such as the ethyl derivative in the mercaptan function, areuseful.

Of the higher saturated aliphatic alcohols, preferred are cetyl alcoholand myricyl alcohol, but for the aim of the present invention, thehigher unsaturated alcohols with one or two double bonds are especiallyimportant, such as especially those contained in many essential oils andwith affinity to terpene, such as citronellol, geraniol, nerol,nerolidol, linalool, farnesol, and phytol. Of the unsaturated loweralcohols, it is necessary to consider allyl alcohol and propargylalcohol. Of the araliphatic alcohols, preferred are those with only onebenzene residue and in which the aliphatic chain has a maximum of 4carbon atoms, where the benzene residue can be substituted by between 1and 3 methyl or hydroxyl groups or by halogen atoms, especially bychlorine, bromine and iodine, and in which the aliphatic chain may besubstituted by one or more functions chosen from the group containingfree amine groups or mono- or dimethylated, or by pyrrolidine orpiperidine groups. Of these alcohols, most preferred are benzyl alcoholand ethyl alcohol.

The alcohols of the cycloaliphatic or aliphatic-cycloaliphatic seriesmay derive from mono- or polycyclic hydrocarbons, may preferably have amaximum of 14 carbon atoms, may be unsubstituted, and may contain one ormore substituents, such as those mentioned above for the aliphaticalcohols. Of the alcohols derived from cyclic monoannular hydrocarbons,preferred are those with a maximum of 12 carbon atoms, the rings withpreferably between 5 and 7 carbon atoms, which may be substituted, forexample, with between one and three lower alkyl groups, such as methyl,ethyl, propyl or isopropyl groups. As specific alcohols of this group,the following are most preferred: cyclohexanol, cyclohexanediol,1,2,3-cyclohexanetriol, and 1,3,5-cyclohexanetriol (phloroglucitol),inositol, and the alcohols which derive from p-methane such ascarvomenthol, menthol, and α-γ terpineol, 1-terpineol, 4-terpineol andpiperitol, or the mixture of these alcohols known as "terpineol", 1,4-and 1,8-terpin. Of the alcohols which derive from hydrocarbons withcondensed rings, such as those of thujane, pinane or comphane, thefollowing are preferred: thujanol, sabinol, pinol hydrate, D- andL-borneol, and D- and L-isoborneol.

Aliphatic-cycloaliphatic polycyclic alcohols to be used for the estersof the present invention include sterols, cholic acids, and steroids,such as sexual hormones and their synthetic analogues, especiallycorticosteroids and their derivatives. It is therefore possible to usecholesterol, dihydrocholesterol, epidihydrocholesterol, coprostanol,epicoprostanol, sitosterol, stigmasterol, ergosterol, cholic acid,deoxycholic acid, lithocholic acid, estriol, estradiol, equilenin,equilin, and their alkylate derivatives, as well as their ethynyl orpropynyl derivatives in position 17, such as 17α-ethynl-estradiol or7α-methyl -17α-ethynyl-estradiol, pregnenolone, pregnanediol,testosterone and its derivatives, such as 17α-methyltestosterone, 1,2-dehydrotestosterone and 17α-methyl-1,2-dehydrotesterone, the alkynylatederivatives in position 17 of testosterone and 1,2-dehydrotestosterone,such as 17α-ethynyltestosterone, 17α-propynyltestosterone, norgestrel,hydroxyprogesterone, corticosterone, deoxycorticosterone,19-nortestosterone, 19-nor-17α-methyltestosterone and19-nor-17α-ethynyltestosterone, antihormones such as cyproterone,cortisone, hydrocortisone, prednisone, prednisolone, fluorocortisone,dexamethasone, betamethasone, paramethasone, flumethasone, fluocinolone,fluprednylidene, clobetasol, beclomethasone, aldosterone,deoxycorticosterone, alfaxolone, alfadolone, and bolasterone. Asesterifying components for the esters of the present invention thefollowing are useful: genins (aglycons) of the cardioactive glucosides,such as digitoxigenin, gitoxigenin, digoxigenin, strophanthidin,tigogenin and saponins.

Other alcohols to be used according to the present invention are thevitamin ones, such as axerophthol, vitamins D₂ and D₃, aneurine,lactoflavine, ascorbic acid, riboflavine, thiamine, and pantothenicacid.

Of the heterocyclic acids, the following can be considered asderivatives of the above-mentioned cycloaliphatic oraliphatic-cycloaliphatic alcohols if their linear or cyclic chains areinterrupted by one or more, for example by between one and threeheteroatoms, for instance chosen from the group formed by --O--, --S--,--N, and --NH--, and in these, there may be one or more unsaturatedbonds, for example double bonds, in particular between one and three,thus including also heterocyclic compounds with aromatic structures. Forexample, the following should be mentioned: furfuryl alcohol, alkaloidsand derivatives such as atropine, scopolamine, cinchonine, 1acinchonidine, quinine, morphine, codeine, nalorphine,N-butylscopoiammonium bromide, ajmaline; phenylethylamines such asephedrine, isoproterenol, epinephrine; phenothiazine drugs such asperphenazine, pipothiazine, carphenazine, homofenazine, acetophenazine,fluophenazine, and N-hydroxyethylpromethazine chloride; thioxanthenedrugs such as flupenthixol and clopenthixol; anticonvulsants such asmeprophendiol; antipsychotics such as opipramol; antiemetics such asoxypendyl; analgesics such as carbetidine and phenoperidine andmethadol; hypnotics such as etodroxizine; anorexics such as benzidroland diphemethoxidine; minor tranquilizers such as hydroxyzine; musclerelaxants such as cinnamedrine, diphylline, mephenesin, methocarbamol,chlorphenesin, 2,2-diethyl-1,3-propanediol, guaifenesin, hydrocilamide;coronary vasodilators such as dipyridamole and oxyfedrine; adrenergicblockers such as propanolol, timolol, pindolol, bupranolol, atenolol,metoprolol, practolol; antineoplastics such as 6-azauridine, cytarabine,floxuridine; antibiotics such as chloramphenicol, thiamphenicol,erythromycin, oleandomycin, and lincomycin; antivirals such asidoxuridine; peripheral vasodilators such as isonicotinyl alcohol;carbonic anhydrase inhibitors such as sulocarbilate; antiasthmatic andantiinflammatories such as tiaramide; and sulfamidics such as2-p-sulfanilonoethanol.

In some cases, hyaluronic acid esters may be of interest where the estergroups derive from two or more therapeutically active hydroxylicsubstances, and naturally all possible variants may be employed.Especially interesting are the substances in which two types ofdifferent ester groups deriving from drugs of a hydroxylic character arepresent, and in which the remaining carboxyl groups are free, salifiedwith metals, or with a base, possibly also the bases being themselvestherapeutically active, for example with the same or similar activity asthat of the esterifying component. In particular, it is possible to usehyaluronic esters deriving on the one hand from an antiinflammatorysteroid, such as one of those mentioned previously, and on the otherhand from a vitamin, from an alkaloid, or from an antibiotic, such asone of those listed.

EXAMPLE 25

Methods of preparing the hyaluronic acid esters of the present invention

Method A:

The esters of hyaluronic acid may be prepared by methods known per sefor the esterification of carboxylic acids, for example by treatment offree hyaluronic acid with the desired alcohols in the presence ofcatalyzing substances, such as strong inorganic acids or ionicexchangers of the acid type, or with an etherifying agent capable ofintroducing the desired alcoholic residue in the presence of inorganicor organic bases. As esterifying agents, it is possible to use thoseknown in literature, such as especially the esters of various inorganicacids or of organic sulphonic acids, such as hydracids, that ishydrocarbyl halogenides, such as methyl or ethyl iodide, or neutralsulphates or hydrocarbyl acids, alfites, carbonates, silicates,phosphites or hydrocarbyl sulfonates, such as methyl benzene orp-toluene-sulfonate or methyl or ethyl chlorosulfonate. The reaction maytake place in a suitable solvent, for example an alcohol, preferablythat corresponding to the alkyl group to be introduced in the carboxylgroup. But the reaction may also take place in non-polar solvents, suchas ketones, ethers, such as dioxane or aprotic solvents, such asdimethyl -sulphoxide. As a base it is possible to use for example ahydrate of an alkaline or alkaline earth metal or magnesium or silveroxide or a basic salt or one of these metals, such as a carbonate, and,of the organic bases, a tertiary azotized base, such as pyridine orcollidine. In the place of the base it is also possible to use an ionicexchanger of the basic type.

Another esterification method employs the metal salts or salts withorganic azotized bases, for example ammonium or ammonium substitutesalts. Preferably, the salts of the alkaline or alkaline earth metalsare used, but also any other metallic salt may be used. The esterifyingagents are also in this case those mentioned above and the same appliesto the solvents. It is preferable to use aprotic solvents, for exampledimethylsulphoxide and dimethylformamide.

In the esters obtained according to this procedure or accordingto theother procedure described hereafter, free carboxylic groups of thepartial esters may be salified, if desired, in a per se known manner.

Method B:

The hyaluronic esters may also be prepared by a method which consists oftreating a quaternary ammonium salt of hyaluronic acid with anetherifying agent, preferably in an aprotic organic solvent.

As organic solvents it is preferable to use aprotic solvents, such asdialkylsulphoxides, dialkylcarboxamides, such as in particular loweralkyl dialkylsulphoxides, especially dimethyl-sulphoxide, and loweralkyl dialkylamides of lower aliphatic acids, such as dimethyl ordiethyl-formamide or dimethyl or diethylacetamide.

Other solvents however are to be considered which are not alwaysaprotic, such as alcohols, ethers, ketones, esters, especially aliphaticor heterocyclic alcohols and ketones with a lower boiling point, such ashexafluoroisopropanol, trifluoroethanol, and N-methylpyrrolidone.

The reaction is effected preferably at a temperature range of betweenabout 0° C. and 100° C., especially between about 25° C. and 75° C., forexample at about 30° C.

The esterification is carried out preferably by adding by degrees theesterifying agent to the above mentioned ammonium salt to one of theabove mentioned solvents, for example to dimethyl-sulphoxide.

As an alkylating agent it is possible to use those mentioned above,especially the hydrocarbyl halogens, for example alkyl halogens. Asstarting quaternary ammonium salts it is preferable to use the lowerammonium tetraalkylates, with alkyl groups preferably between 1 and 6carbon atoms. Mostly, hyaluronate of tetrabutylammonium is used. It ispossible to prepare these quaternary ammonium salts by reacting ametallic salt of hyaluronic acid, preferably one of those mentionedabove, especially sodium or potassium salt, in aqueous solution with asalified sulphonic resin with a quaternary ammonium base.

One variation of the previously described procedure consists in reactinga potassium or sodium salt of hyaluronic acid, suspended in a suitablesolution such as dimethylsulphoxide, with a suitable alkylating agent inthe presence of catalytic quantities of a quaternary ammonium salt, suchas iodide of tetrabutylammonium.

In the partial esters of the invention the non-esterified carboxylicgroups may be kept free or may be salified. For the formation of suchsalts the bases are chosen according to the criterion of these for whichthe product is intended. It is possible to form inorganic salts derivingfrom alkaline metals, such as potassium and especially sodium andammonium, or deriving from alkaline earth metals, such as calcium, ormagnesium or aluminum salts.

Particularly interesting are the salts with organic bases, especiallynitrogenized bases and therefore aliphatic, arylaliphatic,cycloaliphatic or heterocyclic amines.

These ammonia salts may derive from therapeutically acceptable butinactive amines or from amines with therapeutic action. Of the formerthe aliphatic amines above all should be considered, such as mono-, di-and tri-alkylamines with alkyl groups having a maximum of 18 carbonatoms or arylalkylamines with the same number of carbon atoms in thealiphatic part and where aryl means a benzene group possibly substitutedby 1 and 3 methyl groups or halogen atoms or hydroxyl groups. Thebiologically inactive bases for the formation of salts may also becyclic such as monocyclic alkylenamines with cycles of between 4 and 6carbon atoms, possible interrupted in the cycle by heteroatoms chosenfrom the group formed by nitrogen, oxygen and sulfur, such as piperidineor morpholine, and may be substituted for example by aminic orhydroxylic functions, such as aminoethanol, ethylendiamine, ephedrine orcholine.

It is also possible to form the quaternary ammonium salts of the partialesters, for example the salts of tetraalkylammonium with theabove-mentioned number of carbon atoms and preferably salts of such atype in which the fourth alkyl group has between 1 and 4 carbon atoms,for example a methyl group.

Among the biologically active amines whose therapeutic actions may beput to use, are included all the nitrogenized and basic drugs such asthose included in the following groups: alkaloids, peptides,phenothiazines, benzodiazepines, thioxanthenes, hormones, vitamins,anticonvulsants, antipsychotics, antiemetics, anesthetics, hypnotics,anorexics, tranquilizers, muscle relaxants, coronary vasodilators,antineoplastics, antibiotics, antibacterials, antivirals, antimalarials,carbonic anhydrase inhibitors, non-steroid antiinflammatory agents,vasoconstrictors, cholinergic agonists, cholinergic antagonists,adrenergic agonists, adrenergic antagonists, and narcotic antagonists.

All those drugs with basic nitrogenized groups listed in the inventionand regarding the use of the esters may be quoted as examples.

According to a particular aspect of the present invention the hyaluronicesters and their salts may be used as an excellent vehicle fortherapeutically active substances. To this end it is possible to use thetotal esters or the partial esters of the salified partial esters in theremaining carboxylic groups, for example with one of the above-mentionedsubstances therapeutically acceptable but not biologically active, aboveall with alkaline metals, for example sodium. These are theabove-mentioned medicaments made by association containing twocomponents:

Component (1)--a pharmacologically active substance or an association oftwo or more active substances; and

Component (2)--a carrying vehicle comprising a partial or total ester ofhyaluronic acid with an alcohol, or the salts of such partial esterswith an organic or inorganic base, optionally with the addition ofhyaluronic acid or a salt thereof with an inorganic or organic base.

The hyaluronic esters to be used in these medicaments are above allthose in which the esterifying alcohol is not pharmacologically active,for example a simple aliphatic alcohol, as described above. Medicamentsof this type in which the ester also is pharmacologically active, forexample a simple aliphatic alcohol, as described above. Medicaments ofthis type in which the ester also is pharmacologically active are notexcluded from this aspect of the invention, as for example in the caseof one of the esters described above deriving from alcohols withpharmacological action.

In the same way, the invention also includes medicaments of this type inwhich the esters of Component (2) are salified also with therapeuticallyactive bases. These bases maybe the same pharmacologically activesubstances vehicled in the hyaluronic ester, and the mixture in thiscase, as describe below, therefore contains salts of a partial ester ofhyaluronic acid with therapeutically active bases, possibly in thepresence of an excess of active base component (1). The case may on theother hand present itself where the vehicled substance is not of a basicnature, and free carboxylic groups in the hyaluronic ester are stillsalified with therapeutically active bases.

The use of hyaluronic esters as a vehicle allows therefore thepreparation of the medicaments described above, including (1) apharmacologically active substance or an association of two or more ofsuch substances and (2) a hyaluronic ester as described above or one ofits salts. In such medicaments, if partial esters of HA are used, thepossible salification of the uremaining carboxylic groups is carried outpreferably with therapeutically neutral inorganic or organic bases,especially with alkaline metals such as sodium or ammonium. Should theactive substance component (1) or a corresponding association ofsubstances have basic groups, such as for example antibiotics containingamine groups, and if partial esters of hyaluronic acid should be usedwith remaining free carboxylic groups, the corresponding salts areformed between the carboxylic groups and these basic substances. The newmedicaments therefore include in particular partial esters of hyaluronicacid partially and totally salified with pharmacologically activesubstances and of a basic character. As described above, particularlyimportant are the associated medicaments of the type described herein,in which Component (1) is a pharmacologically active substance fortopical use.

The use of hyaluronic esters as a vehicle for drugs to be appliedtopically is particularly useful in ophthalmology where a particularcompatibility is to be observed for the products with the cornealepithelium, and therefore excellent tolerability, without anysensitization effects. Furthermore, when the medicaments areadministered in the form of concentrated solutions with elastic-viscosecharacteristics or in solid form, it is possible to achieve homogenousand stable films which are perfectly transparent and adhesive on thecorneal epithelium, guaranteeing prolonged biovavailability of the drugand therefore representing excellent preparations with a retard effect.

Such ophthalmic medicaments are particularly valuable in the veterinaryfield, considering for example that at present there are no veterinaryspecialties for oculistic use containing chemotherapeutic agents.Indeed, preparations intended for human use are usually used, and thesedo not always guarantee a specific range of action or they do not makeallowances for the particular conditions in which the treatment musttake place. This, for example, is the case in therapy for infectirekerato-conjunctivities, pink eye or IBK, an infection which usuallyaffects cattle, sheep and goats. Presumably for these three speciesthere are specific etiological factors and more particularly: in cattlethe main microorganism involved seems to be Moraxella boris (even thoughother agents of a viral origin should not be excluded, such as forexample Rinotracheitis virus, in sheep Micoplasma, Rickettsiae andClamidiae, and in goats Rickettesiae). The disease manifests itself inacute form and tends to spread rapidly: in the initial stages thesymptomatology is characterized by blepharospasm and excessivelachrymation, followed by purulent exudate, conjunctivities andkeratitis, often accompanied by fever, loss of appetite and milkproduction. Particularly serious are the corneal lesions which in thefinal stages may even cause perforation of the cornea itself. Theclinical progress of the disease varies from a few days to severalweeks.

A vast selection of chemotherapeutic agents are used for treatment,administered both topically (often in association with steroidantiinflammatory agents), and systemic, including: tetracyclines, suchas oxytetracycline, penicillins, such as cloxacillin andbenzylpenicillin, sulfonamides, polymyxin B (associated with miconazoleand prednisolone), chloramphenicol, tylosin and ckloromycetin. Topicaltreatment of the disease, despite its apparent simplicity, is still anunsolved problem, since with the oculistic preparations used so far, ithas not been possible for one reason or another, to obtaintherapeutically efficient concentrations of antibiotics or sulphamidesin the lachrymal secretion. This is quite understandable in the case ofsolutions, considering the mainly inclined position of the head in theseanimals, but the same is also true of semisolid medicaments, as thecommonly used excipients do not possess the necessary qualities ofadhesiveness to the corneal surface, since they do not usually have ahigh enough concentration of active substance and cannot achieve perfectdistribution of the same (i.e., there is a presence of a distributiongradient). These defects of conventional colliriums in ophthalmic usehave for example been described by Slatter et al., Austr. Vet. J., 1982,59(3), pp. 69-72.

With the esters of the present invention these difficulties can beovercome. The presence of the hyaluronic acid ester as a vehicle forophthalmic drugs in fact allows the formulation of excellentpreparations with no concentration gradients of the active substance andthey are therefore perfectly homogenous, with perfect transparency andexcellent adhesiveness to the corneal epithelium, with no sensitizationeffects, with excellent vehicling of the active substance and possibly aretard effect.

The above-mentioned properties of the medicaments may of course beexploited also in fields other than ophthalmology. They may be used indermatology and in diseases of the mucous membranes, for example in themouth. Furthermore, they may be used to obtain a systemic effect due tothe effect of transcutaneous absorption, such as in suppositories,. Allthese applications are possible both in human and veterinary medicine.In human medicine the new medicaments are particularly suitable for usein pediatrics. The present invention includes therefore in particularany of these therapeutic applications.

For the sake of brevity, from now on when the active substance ofcomponent (1) according to the invention is mentioned, it is to beunderstood to also include the association of one or more activesubstances.

The component (1) described above may first of all be defined in regardto its use in the various fields of therapy, starting with thedistinction between human and veterinary medicine, and then specifyingthe various sectors of application with regard to the organs or tissuesto be treated, such as, with reference to topical use, ophthalmology,dermatology, otorhinolaryngology, gynecology, angiology, neurology, orany type of pathology of internal organs which may be treated withtopical applications, for example with rectal applications.

The vehicling action of the hyaluronic esters also applies to associatedmedicaments of the type mentioned above in which the active substanceacts not only topically or by nasal or rectal absorption, for example bynasal sprays or preparations for inhalation for the oral cavity or thepharynx, but also by oral or parenteral route, for example byintramuscular, subcutaneous or intravenous route, as it favorsabsorption of the drug into the application site. The medicaments cantherefore be applied, apart from in the fields already mentioned, inpractically all sectors of medicine, such as internal medicine, forexample in pathologies of the cardiovascular system, in infections ofthe respiratory system, the digestive system, the renal system, indiseases of an endocrinological nature, in oncology, in psychiatry,etc., and may also be classified therefore from the point of view oftheir specific action, being perhaps anesthetics, analgesics,antiinflammatories, wound healers, antimicrobics, adrenergic agonistsand antagonists, cytostatics, antirheumatics, antihypertensives,diuretics, sexual hormones, immunostimulants and immunosuppressants, forexample, one of the drugs having the activity already described for thetherapeutically active alcohols to be used as esterifying component, orfor the therapeutically active bases used for the salification of thefree carboxylic groups.

Component (1) of the above mentioned medicaments may also be, accordingto the invention, an association of two or more active substances, ascontained in many known medicaments.

Regarding the field of ophthalmology, the indications may be forexample: the miotic, antiinflammatory, wound healing and antimicrobialeffects.

Examples of pharmacologically active substances to be used in ophthalmicmedicaments according to the invention are: basic and non-basicantibiotics such as aminoglycosides, macrolides, tetracyclines andpeptides, such as gentamycin, neomycin, streptomycin,dihydrostreptomycin, kanamycin, amikacin, tobramycin, spectinomycin,erythromycin, oleandomycin, carbomycin, spiramycin, oxytetracycline,rolitetracycline, bacitracin, polymyxin B, gramicidin, colistin,chloramphenicol, lincomycin, vancomycin, novobiocin, ristocetin,clindamycin, amphotericin B, griseofulvin, nystatin, and possibly theirsalts such as sulfates or nitrates, or associations of the same betweenthemselves or with other active ingredients, such as those mentionedbelow.

Other ophthalmic drugs to be used to advantage according to the presentinvention are: other antiinfectives such as diethylcarbamazine,mebendazole, sulfamedics such as sulfacetamide, sulfadiazine,sulfisoxazole, antivirals and antitumorals such as iododeoxyuridine,adenine arabinoside, trifluorothymidine, acyclovir, ethyldeoxyuridine,bromovinyldeoxyuridine, 5-iodo-5'-amino-2',5'-dideoxyuridine; steroidantiinflammatories, such as dexamethasone, hydrocortisone, prednisolone,fluorometholone, medrysone and possibly their esters, for examplephosphoric acid; non-steroid antiinflammatories such as indomethacin,oxyphenbutazone, flurbiprofen; wound healers such as epidermal growthfactor, EGF; local anesthetics, such as Benoxinate, proparacaine andpossibly their salts; cholinergic agonists such as pilocarpine,methcholine, carbomylcholine, aceclidine, physostigmine, neostigmine,demecarium and possibly their salts; cholinergic antagonist drugs suchas atropine and their salts; adrenergic agonist drugs such asnoradrenaline, adrenaline, naphazoline, methoxamine and possibly theirsalts; adrenergic antagonist drugs such as propanolol, timolol,pindolol, bupranolol, atenolol, metoprolol, oxprenolol, practolol,butoxamine, sotalol, butathrin, labetolol and possibly their salts.

Examples of the active substances to be used alone or in associationamong themselves or with other active principles in dermatology are:therapeutic agents such as antiinfective agents, antibiotics,antimicrobials, antiinflammatory, cytostatic, cytotoxic, antiviral,anesthetic agents, and prophylactic agents, such as sun screens,deodorants, antiseptics and disinfectants. Of the antibiotics,particularly important are: erythromycin, bacitracin, gentamicin,neomycin, aureomicin, gramicidin and their associations; of theantibacterials and disinfectants: nitroflurzone, mafenide,chlorhexidine, and derivatives of 8-hydroxyquinoline and possibly theirsalts; of the antiinflammatory agents, above all the corticosteroidssuch as prednisolone, dexamethazone, flumethasone, clobetasol,triamcinolone acetonide, betamethasone and their esters, such asvalerates, benzoates, dipropionates; of the cytotoxic group:fluorouracil, methotrexate, podophyllin; of the anesthetics, dibucaine,lidocaine, and benzocaine.

This list of course only gives some examples and any other agentsdescribed in the literature may be used.

As associations of drugs to be used in dermatology, the variousantibiotics should be mentioned, such as erythromycin, gentamycin,neomycin, gramicidin, polymyxin B, among themselves, or associations ofthese antibiotics with antiinflammatory agents, for examplecorticosteroids, for example hydrocortisone+neomycin,hydrocortisone+neomycin+polymyxin B+gramicidin, dexamethasone+neomycin,fluorometholone+neomycin, prednisolone+neomycin,triamcinolone+neomycin+gramicidin+nystatin, or any other associationused in conventional preparations for dermatology.

The associations of various active substances are not of course limitedto this field, but in each of the above-mentioned areas of medicine itis possible to use associations similar to those already in use for theknown pharmaceutical preparations of the art.

In the above case of the use of a component (1) of a basic character,the salts which are formed with a partial hyaluronic ester (since thelatter is used to excess) may be of various types, that is, all theremaining carboxylic groups may be salified or only an aliquot part,thereby producing esters-acid salts, or esters-neutral salts. The numberof acid groups which are to be kept free may be of importance for thepreparation of medicaments with a particular pH. Vice versa, it ispossible to use an excess of basic component (1), in which case all thecarboxylic groups available in the hyaluronic ester are salified withthe base.

According to a particular aspect of the invention it is possible toprepare the medicaments of this type starting from previously isolatedand possibly purified salts, in their solid anhydrous state, asamorphous powders, which form an aqueous solution on contact with thetissue to be treated, characterized by viscosity and elastic properties.These qualities are maintained even at stronger dilutions and it ispossible therefore to use, in the place of the above-mentioned anhydroussalts, more or less concentrated solutions in water or saline, possiblywith the addition of other excipients or additives, such as for exampleother mineral salts to regulate the pH and osmotic pressure. It is ofcourse possible to use the salts also for the preparation of gels,inserts, creams or ointments, containing also other excipients oringredients used in traditional formulations of these pharmaceuticalpreparations.

According to the invention however, the medicaments containing thehyaluronic esters or their salts with therapeutically active or inactivesubstances as a vehicle are used alone (except possibly with an aqueoussolvent). Also included in the invention are the mixtures obtainablefrom all the types of medicaments described here, mixtures of the samemedicaments, and also possibly mixtures of the hyaluronic acid esterswith free hyaluronic acid or mixtures of their salts, for example sodiumsalts.

Component (1) according to the invention may also be associations ormixtures of two or more such drugs and possibly also with otherprinciples. For example, in ophthalmology, a drug may be associated withan antibiotic or antiphlogistic substance and a vasoconstrictor or withseveral antibiotics, one or more antiphlogistic substances, or with oneor more antibiotics, a mydiatric or a miotic or wound healing orantiallergic agent, etc. For example the following associations ofophthamalic drugs may be used: kanamycin+phenylephrine+dexamethasonephosphate; kanamycin+betamethasone phosphate+phenylephrine, or similarassociations with other antibiotics used in ophthalmology, such asrolitetracycline, neomycin, gentamicin, and tetracycline.

If in the place of just one active substance component (1) associationsof active substances are used, such as those mentioned above, the saltsof the basic active substances and the partial ester of hyaluronic acidmay be mixed salts of one or more of such basic substances or possiblymixed salts of this type with a certain number of other acid groups ofthe polysaccharides salified with metals or bases mentioned above. Forexample, it is possible to prepare salts of a partial ester ofhyaluronic acid or of one of the molecular fractions Hyalastine orHyalectin with a pharmacologically inactive alcohol, for example a loweralkanol and with a certain percentage of salified acid groups with theantibiotic kanamycin, another percentage of carboxylic groups salifiedwith the vasoconstrictor phenylephrine, and a remaining percentage ofacid groups may be, for example, free or salified with sodium or one ofthe other above-mentioned metals. It is also possible to mix this typeof mixed salt with free hyaluronic acid or its fractions or theirmetallic salts, as indicated above-for the medicaments containing saltsof one single active substance with the aforementioned polysaccharideesters.

Of the examples discussed for ophthalmology and dermatology it ispossible to understand by analogy which medicaments according to thepresent invention are to be used in the above-mentioned fields ofmedicine, such as for example in otorhinolaryngology, odontoiogy or ininternal medicine, for example in endocrinology. Such preparations may,therefore, be for example antiinflammatories, vasoconstrictors, orvasocompressors such as those already mentioned for ophthalmology,vitamins, antibiotics, such as those mentioned above, hormones,chemiotherapics, antibacterials, etc., also as mentioned above for usein dermatology.

The associated medicaments of a hyaluronic ester with apharmacologically active substance may contain other pharmaceuticalvehicles, such as those mentioned below for the pharmaceuticalpreparations containing only hyaluronic esters. However, it ispreferable to use medicaments containing an association of components(1) and (2), with component (2) as the sole vehicle (apart from apossible solvent such as an aqueous solvent).

Of the medicaments of the invention the following are of particularimportance, according to each case, those with a degree of aciditysuitable for the environment to which they are to be applied, that iswith a physiologically tolerable pH. The adjustment of the for examplein the above-mentioned salts of the partial ester of hyaluronic acidwith a basic active substance, may be done by suitably regulating thequantities of polysaccharide, of its salts and of the basic substanceitself. Thus, for example, if the acidity of a salt of the partial esterof hyaluronic acid with a basic substance is too high, the excess offree acid groups cans be neutralized with the above-mentioned inorganicbases, for example with the hydrate of sodium or potassium or ammonium.

METHOD B

The hyaluronic esters of the present invention may, however, be preparedto advantage according to a second method which may be generally appliedto the preparation of carboxylic esters of acidic polysaccharides withcarboxyl groups. This method consists of treating a quaternary ammoniumsalt of an acidic polysaccharide containing carboxyl groups with anetherifying agent, preferably in an aprotic organic solvent.

As organic solvents it is preferable to use aprotic solvents such asdialkylsulphoxides, dialkylcarboxamides, such as in particular loweralkyl diatkylsulphoxides, especially dimethylsulphoxide, and lower alkyldialkylamides of lower aliphatic acids, such as dimethyl ordiethylformamide or dimethyl or diethylacetamide.

Other solvents however are to be considered which are not alwaysaprotic, such as alcohols, ethers, ketones, esters, especially aliphaticor heterocyclic alcohols and ketones with a lower boiling point, such ashexafluoroisopropanol, trifluoroethanol, and N-methytpyrrolidone.

The reaction is effected preferably in a temperature range of betweenabout 0° C. and 100° C., especially between about 25° C. and 75° C., forexample at about 30° C.

The esterification is carried out preferably by adding by degrees theesterifying agent to the above-mentioned ammonium salt in one of theabove-mentioned solvents, for example to dimethylsulphoxide.

As an alkylating agent it is possible to use those mentioned above,especially the hydrocarbyl halogens, for example alkyl halogens. Asstarting quaternary ammonium salts it is preferably to use the lowerammonium tetraalkylates, with alkyl groups preferably between 1 and 6carbon atoms. Mostly, tetrabutylammonium hyaluronate is used. It ispossible to prepare these quaternary ammonium salts by reacting ametallic salt of an acidic polysaccharide, preferably one of thosementioned above, especially a sodium or potassium salt, in aqueoussolution with a salified sulphonic resin with aa quaternary ammoniumbase.

The tetraalkylammonium salt of the acidic polysaccharide can be obtainedby freeze drying the eluate. The tetraalkylammonium salts of acidicpolysaccharides used as starting compounds of the present procedure canderive from inferior alkyls, especially alkyls with between 1 and 6carbon atoms. Surprisingly, such salts have proved to be soluble in theabove-mentioned organic solvents, and for this reason the esterificationof acidic polysaccharides according to procedure B is particularly easyand gives generous yields. It is therefore only by using this kind ofprocedure that one can exactly dose the number of carboxylic groups ofacidic polysaccharide which are to be esterified.

One variation of previously described procedure B consists in reactingpotassium salt or acidic polysaccharide sodium, suspended in a suitablesolution such as dimethylsulphoxide, with a suitable alkylating agent inthe presence of catalytic quantities of a quaternary ammonium salt, suchas iodide of tetrabutylammonium.

The salification of HA with the above metals, for the preparation ofstarting salts for the particular esterification procedure of thepresent invention described above, is performed in a per se knownmanner, for example by reacting HA with the calculated base quantity,for example with alkaline hydrates or with basic salts of such metals,such as carbonates or bicarbonates.

In the partial esters of the present invention it is possible to salifyall the remaining carboxylic groups or only part of them, dosing thebase quantities so as to obtain the desired stoichiometric degree ofsalification. With the correct degree of salification it is possible toobtain esters with a wide range of different dissociation constants andwhich therefore give the desired pH in solution or in situ at the timeof therapeutic application.

Of the products of the present invention, of particular importance arethe esters and their salts described above and those described in thefollowing illustrative Examples.

Preparation of Benzyl and Ethyl Esters of Hyaluronic Acid EXAMPLE 26

Preparation of the total benzyl ester (HYAFF11) of hyaluronic acid

12.4 g of HA tetrabutylammonium salt with a molecular weight of 170,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°. 4.5 g (25 m.Eq.) of benzyl bromide and 0.2g of tetrabutylammonium iodide are added, and the solution is kept for12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty-four hours at 30°.

9 g of the benzyl ester product in the title are obtained. Quantitativedetermination of the ester groups is carried out according to the methoddescribed on pages 169-172 of Siggia S. and Hanna J. G., "QuantitativeOrganic Analysis Via Functional Groups," 4th edition, John Wiley andSons.

Alternatively, 3 g of the potassium salt of HA with a molecular weightof 162,000 are suspended in 200 ml of dimethylsulfoxide; 120 mg oftetrabutylammonium iodide and 2.4 g of benzyl bromide are added.

The suspension is kept in agitation for 48 hours at 30°. The resultingmixture is slowly poured into 1,000 ml of ethyl acetate under constantagitation. A precipitate is formed which is filtered and washed fourtimes with 150 ml of ethyl acetate and finally vacuum dried for twentyfour hours at 30°.

3.1 g of the benzyl ester product in the title are obtained.Quantitative determination of the ester groups is carried out accordingto the method described on pages 169-172 of Siggia S. and Hanna J. G.,"Quantitative Organic Analysis Via Functional Groups," 4th edition, JohnWiley and Sons.

EXAMPLE 27

Preparation of partial benzyl esters (HYAFF 11 p10, p25, p50, and p75)of hyaluronic acid

The partial benzyl esters of hyaluronic acid, HYAFF 11 p10, p25, p50,and p75, can be prepared as described in Method B, supra. Theesterification can be carried out by adding by degrees the esterifyingagent to the quaternary ammonium salt of hyaluronic acid treated with anetherifying agent in an appropriate organic solvent.

The salification of hyaluronic acid for the preparation of startingsalts for esterification and the salification of the remaining carboxylgroups in the partial benzyl esters is also described in Method B.

EXAMPLE 28

Preparation of the ethyl ester (HYAFF 7) of hyaluronic acid

12.4 g of HA tetrabutylammonium salt with a molecular weight of 85,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°. 3.3 g (21.2 m.Eq.) of ethyl iodide areadded and the solution is kept for 12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty-four hours at 30°.

8 g of the ethyl ester product in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030, (1961).

EXAMPLE 29

Preparation of the (partial) cortisone ester (C₂₁) of hyaluronic acid(HA)-20% of esterified carboxylic groups-80% of salified carboxylicgroups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 105,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.850 g (2 m.Eq) of21-bromo-4-pregnene-17α-ol-3,11,20-trione are added and the resultingsolution is kept for 24 hours at 30°.

A solution containing 100 ml of water and 5 g of sodium chloride isadded and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which filteredand washed three times with 100 ml of acetone/water 5:1 and three timeswith acetone and finally vacuum dried for eight hours at 30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.5 g of thepartial cortisone ester compound in the title are obtained. Quantitativedetermination of cortisone, after mild alkaline hydrolysis with ahydroalcoholic solution of Na₂ CO₃ and extraction with chloroform, iscarried out according to British Pharmacopea, 1980, P. 127.

EXAMPLE 30

Preparation of the (partial) hydrocortisone ester (C₂₁) of hyaluronicacid (HA)-20% of esterified carboxylic groups-80% of salified carboxylicgroups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 80,000corresponding to 20 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.850 g (2 m.Eq of21-bromo-4-pregnene-11β,17α-diol-3,20-dione are added and the resultingsolution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.4 g of thepartial hydrocortisone ester compound in the title are obtained.Quantitative determination of hydrocortisone, after mild alkalinehydrolysis with hydroalcoholic solution of Na₂ --CO₃ and extraction withchloroform, is carried out according to British Pharmacopea, 1980, p.224.

EXAMPLE 31

Preparation of the (partial) fluorocortisone ester (C₂₁) of hyaluronicacid (HA)-20% of esterified carboxylic groups-80% of salified carboxylicgroups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 80,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.89 g (2 m.Eq) of9-fluoro-21-bromo-4-pregnene-11β,17α-diol-3,20-dione are added and theresulting solution is kept for 12 hours at 30°.

A solution is then added containing 62 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours: at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.6 g of thepartial fluorocortisone compound in the title are obtained. Quantitativedetermination of fluorocortisone, after mild alkaline hydrolysis withhydroalcoholic solution of Na₂ CO₃ and extraction with chloroform, iscarried out according to British Pharmacopea, 1980, p. 196.

EXAMPLE 32

Preparation of the (partial) desoxycorticosterone ester (C₂₁) ofhyaluronic acid (HA)-20% of esterified carboxylic groups-80% of salifiedcarboxylic groups (Na)

6.21 g of HA tetrabutylammonium salt with a molecular weight of 105,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.661 g (2 m.Eq) of21-bromo-4-pregnene-3,20-dione are added and the resulting solution iskept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.5 g of thepartial desoxycorticosterone ester compound in the title are obtained.Quantitative determination of desoxycorticosterone, after mild alkalinehydrolysis with hydroalcoholic solution of Na₂ CO₃ and extraction withchloroform, is carried out according to British Pharmacopea, 1980, p.137.

EXAMPLE 33

Preparation of the (mixed) ethanol and cortisone ester (C₂₁) Ofhyaluronic acid (HA)-80% of the carboxylic groups esterified withethanol-20% of the carboxylic groups esterified with cortisone (C₂₁)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 1.25 g (8 m.Eq) of ethyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

0.85 g (2 m.Eq) of 21-bromo-4-pregnene-17a-ol-3,11,20-trione are addedand the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which is thenfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the mixed ethanol and cortisone ester compound in the title areobtained. Quantitative determination of cortisone, after mild alkalinehydrolysis with hydroalcoholic solution of Na₂ CO₃ and extraction withchloroform, is carried out according to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 34

Preparation of the (mixed) ethanol and hydrocortisone ester (C₂₁) ofhyaluronic acid (HA)-80% of carboxylic groups esterified withethanol-20% of carboxylic groups esterified with hydrocortisone (C₂₁)

6.2g of HA tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 1.25 g (8 m.Eq) of ethyl iodide are addedand the solution is kept at 30° for 12 hours.

0.85 g (2 m.Eq) of 21-bromo-4-pregnene-11β, 17α-diol-3,20-dione areadded and the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30° C.

4.6 g of the mixed ethanol and hydrocortisone ester compound in thetitle are obtained. Quantitative determination of hydrocortisone, aftermild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ andextraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030(1961).

EXAMPLE 35

Preparation of the (mixed) ethanol and fluorocortisone ester (C₂₁) ofhyaluronic acid (HA)-80% of carboxylic groups esterified withethanol-20% of carboxylic groups 5 esterified with fluorocortisone C₂₁

6.2g of HA tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 1.25 g (8 m.Eq) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.89 g (2 m. Eg.) of 9β-fluoro-21-bromo-4-pregnene-11β,17α-diol-3,20-dione are added and the solution is kept for 24 hours at30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30° C.

4.6 g of the mixed ethanol and fluorocortisone ester compound featuredin the title are obtained. Quantitative determination offluorocortisone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030(1961).

EXAMPLE 36

Preparation of the (mixed) ethanol and desoxycorticosterone ester (C₂₁)of hyaluronic acid (HA)-80% of carboxylic groups esterified withethanol-20% of carboxylic groups esterified with desoxycorticosterone(C₂₁)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 1.25 g (8 m.Eq) of ethyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

0.661 g (2 m.Eq ) of 21-bromo-4-pregnene-3,20-dione are added and thesolution is kept for 24 hours at 30°. A solution is then addedcontaining 100 ml of water and 5 g of sodium chloride and the resultingmixture is slowly poured into 2,000 ml of acetone under constantagitation. A precipitate is formed which is filtered and washed threetimes with 100 ml of acetone/water 5:1 and three times with 100 ml ofacetone and finally vacuum dried for eight hours at 30°.

4.6 g of the mixed ethanol and desoxycorticosterone ester compound inthe title are obtained. Quantitative determination ofdesoxycorticosterone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 37

Preparation of the (partial and mixed) ethanol and desoxycorticosteroneester of hyaluronic acid IHA)-40% of carboxylic groups esterified withdesoxycorticosterone (C₂₁)-40% of salified carboxylic groups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq of a monomeric unit are 'solubilized in 310 mlof dimethylsulfoxide at 25°. 0.62 g (4 m.Eq) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq) of 21-bromo-4-pregnene-3,20-dione are added and thesolution is kept for 24 hours at 30°. A solution is then addedcontaining 100 ml of water and 5 g of sodium chloride and the resultingmixture is slowly poured into 2,000 ml of acetone under constantagitation. A precipitate is formed which is filtered and washed threetimes with 100 ml of acetone/water 5:1 and three times with 100 ml ofacetone and finally vacuum dried for eight hours at 30°.

4.5 g of the partial and mixed ethanol and desoxycorticosterone estercompound in the title are obtained. Quantitative determination ofdesoxycorticosterone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 38

Preparation of the (partial and mixed) ethanol and cortisone ester ofhyaluronic acid (HA)-40% of carboxylic groups esterified withethanol-20% of carboxylic groups esterified with cortisone (C₂₁)-40% ofsalified carboxylic groups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.62 g (4 m.Eq) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq) of 21-bromo-4-pregnene-17α-ol -3,11,20-trione are addedand the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.5 g of the partial and mixed ethanol and cortisone compound in thetitle are obtained. Quantitative determination of cortisone, after mildalkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ andextraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 39

Preparation of the (partial and mixed) ethanol and hydrocortisone ester(C₂₁) of hyaluronic acid (HA)-40% of carboxylic groups esterified withethanol-20% of carboxylic groups esterified with hydrocortisone (C₂₁)-40% of salified carboxylic groups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.62 g (4 m.Eq) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq) of 21-bromo-4-pregnene-11β,17α-diol-3,20-dione are addedand the solution is kept for 24 hours at 30°.

A solution is then added containing 200 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.5 g of the partial and mixed ethanol and hydrocortisone ester compoundin the title are obtained. Quantitative determination of hydrocortisone,after mild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃and extraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 40

Preparation of the (partial and mixed) ethanol and fluorocortisoneesters (C₂₁) of hyaluronic acid (HA)-40% of carboxylic groups esterifiedwith ethanol-20% of carboxylic groups esterified with fluorocortisone(C₂₁) -40% of salified carboxylic groups (Na)

6.2 g of HA tetrabutylammonium salt with a molecular weight of 65,000corresponding to 20 m.Eq of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°. 0.62 g (4 m.Eq) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.89 g (2 m.Eq) of 9β-fluoro-21-bromo-4-pregnene -11β,17α-diol-3,20-dione are added and the solution is kept for 24 hours at30°.

15 A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml ethyl acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the partial and mixed ethanol and fluorocortisone ester in thetitle are obtained. Quantitative determination of fluorocortisone, aftermild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ andextraction with chloroform, is carried cut according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas, Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 41

Preparation of the streptomycin salt of hyaluronic acid (HA) partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with streptomycin

243 mg of streptomycin sulphate (1 m.Eq) are solubilized in 20 ml ofwater. The solution is eluted in a thermostatic column at 5° containing2 ml of quaternary ammonium resin (Dowex 1×8) in the OH form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HA and 25% sodium salt (corresponding to 1m.Eq of a monomeric unit relative to the non-esterified carboxyl) aresolubilized in 400 ml of water. The solution is eluted in a thermostaticcolumn at 20° and containing 2 ml of sulphonic resin (Dowex 50×8) in theH⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofstreptomycin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.7 g.

Microbiological determination on B. subtilis ATCC 6633 in comparisonwith streptomycin standard shows a content of 10.9% by weight ofstreptomycin base, corresponding to the theoretically calculatedcontent.

EXAMPLE 42

Preparation of the erythromycin salt of hyaluronic acid (HA) partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with erythromycin

1.6 g of a 75% ethyl ester of HA and sodium salt at 25% (correspondingto m.Eq of a monomeric unit relative to the non-esterified carboxyl aresolubilized in 400 ml of water. The solution is eluted in a thermostaticcolumn at 20° containing 2 ml of sulfonic resin (Dowex 50×8) in the H⁺form.

To the sodium-free eluate are added 734 mg of erythromycin base (1m.Eq). The resulting solution is instantly frozen and freeze-dried.Yield: 2.1 g.

Microbiological determination on S. aureus ATCC 6538 in comparison tostandard erythromycin shows a content of 31.7% by weight of erythromycinbase, corresponding to the theoretically calculated weight.

EXAMPLE 43

Preparation of the neomycin salt of hyaluronic acid partially esterifiedwith ethanol-75% of carboxylic groups esterified with ethanol-25% ofcarboxylic groups salified with neomycin

152 mg of neomycin sulfate (1 m.Eq) are solubilized in 20 ml of water.The solution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowex 1×8) in the OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HA and sodium salt at 25% (correspondingto 1 m. Eg. of monomeric unit relative to the non-esterified carboxyl)are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in the H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofneomycin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.65 g.

Microbiological determination carried out on S. aureus ATCC 6538 incomparison to standard neomycin shows a content of 6.1% by weight ofneomycin base, corresponding to the theoretically calculated value.

EXAMPLE 44

Preparation of the gentamicin salt of hyaluronic acid (HA) partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with gentamicin

145 mg of gentamicin sulfate are solubilized in 10 ml of water. Thesolution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowex 1×8) in the OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HA and sodium salt at 25% (correspondingto 1 m.Eq of a monomeric unit relative to the non-esterified carboxyl)are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in the H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofgentamicin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.7 g.

Microbiological determination carried out on S. epidermidus ATCC 12228in comparison to standard gentamicin shows a content of 6.50% by weightof gentamicin base, corresponding to the theoretically calculated value.

EXAMPLE 45

Preparation of the amikacin salt of hyaluronic acid (HA) partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with amikacin

147 mg of amikacin (1 m.Eq) are solubilized in 20 ml of water.

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto 1 m.Eq of a monomeric unit relative to the non-esterified carboxyl)are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in the H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofamikacin base. The resulting solution is instantly frozen and freezedried. Yield: 1.70 g.

Microbiological determination carried out on S. aureus ATCC 29737 incomparison to standard amikacin shows a content of 8.5% by weight ofamikacin base, corresponding to the theoretically calculated value.

EXAMPLE 46

Preparation of the kanamycin salt of hyaluronic acid (HA) partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with kanamycin

146 mg of kanamycin sulfate (1 m.Eq) are solubilized in 20 ml of water.The solution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowex 1×8) in the OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HA and sodium salt at 25% (correspondingto 1 m.Eq of a monomeric unit relative to the non-esterified carboxyl)are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in the H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofkanamycin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.5 g.

Microbiological determination carried out on B. subtilis ATCC 6633 incomparison to standard kanamycin shows a content of 7% by weight ofkanamycin base, corresponding to the theoretically calculated value.

EXAMPLE 47

Preparation of the pilocarpine salt of hyaluronic acid (HA) Partiallyesterified with ethanol-75% of carboxylic groups esterified withethanol-25% of carboxylic groups salified with pilocarpine

245 mg of pilocarpine hydrochloride (1 m.Eq) are solubilized in 20 ml ofwater. The solution is eluted in a thermostatic column at 5° containing2 ml of quaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The chloride-free eluate is gathered in a thermostatic container at 5°.

1.6 g of a 75% ethyl ester of HA and sodium salt at 25% (correspondingto 1 m.Eq of a monomeric unit relative to the non-esterified carboxyl)are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in the H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofpilocarpine base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.89 g.

EXAMPLE 48

Preparation of the pilocarpine salt of hyaluronic acid (HA) partiallyesterified with n-propanol-85% of carboxylic groups esterified withn-propanol-15% of carboxylic groups salified with pilocarpine

245 mg of pilocarpine hydrochloride (1 m.Eq) are solubilized in 10 ml ofwater. The solution is eluted in a thermostatic column at 5° containing2 ml of quaternary ammonium resin (Dowex 1×8) in the OH⁻ form.

The chloride-free eluate is gathered in a thermostatic container at 5°.

4.1 g of the 85% propyl ester of HA and tetrabutylammonium salt at 15%(corresponding to 1 m.Eq of a monomeric unit relative to thenonesterified carboxyl) are solubilized in 100 ml of dimethylsulfoxide.The solution is eluted in a thermostatic column at 20° containing 2 mlof damp sulfonic resin (Dowex 50×8) in the H⁺ form.

The eluate is gathered under agitation in the solution of pilocarpinebase. The resulting solution is precipitated with ethyl acetate (600ml).

The precipitate is filtered and washed four times with 200 ml of ethylacetate and finally vacuum dried for 24 hours at 30°. 3.5 g of thecompound featured in the title are obtained.

The following examples illustrate the preparation of films byevaporation of solvent from mixtures of hyaluronic acid ester withbenzyl alcohol (25% esterification, HYAFF11 p25) dissolved in DMSO andpolymers soluble in DMSO.

The solution of HYAFF11 p25 in DMSO is prepared as follows: 100 mg ofHYAFF11 p25 are dissolved in distilled water: DMSO, 1:1, while beingshaken at room temperature for 30 minutes. The water is then substitutedwith DMSO by adding the second solvent to the solution and heating it to90° C. until all the water has evaporated. Lastly, the solution isbrought to a final volume of 10 ml with DMSO. A 1% solution of HYAFF11p25 is thus obtained, and will be referred to as solution A in examples49-55.

EXAMPLE 49

Preparation of a film containing HYAFF11p25 and polyvinyl alcohol (PVA)

100 mg of PVA are dissolved in DMSO while being shaken at a temperatureof 100° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PVA is thus obtained, which will be referred to assolution B.

Solution A is slowly added to solution B while being continuously shakenat a temperature of 100° C. The resulting solution is shaken for onehour to allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11p25/PVA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 50

Preparation of a film containing HYAFF11p25 and Clarene L6, Solvay (C1L6), ethylene-vinyl alcohol copolymer with a low ethylene content (29mole %)

100 mg of C1 L6 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 L6 is thus obtained, which will bereferred to as solution C.

Solution A is slowly added to solution C while being continuously shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11p25/C1 L6 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced into a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 51

Preparation of a film containing HYAFF11p25 and Clarene P10, Solvay (C1P10), ethylene-vinyl alcohol copolymer with a medium ethylene content(36 mole %)

100 mg of C1 P10 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 P10 is thus obtained, which will bereferred to as solution D.

Solution A is slowly added to solution D while being shaken continuouslyat a temperature of 80° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11p25/C1 P10 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced into a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 52

Preparation of a film containing HYAFF11p25 and Clarene R20, Solvay (C1R20), ethylene-vinyl alcohol copolymer with a high ethylene content (40mole %)

100 mg of C1 R20 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 R20 is thus obtained, which will bereferred to as solution E.

Solution A is slowly added to solution E while being continuously shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution containing the mixture of HYAFF11 p25/C1 R20 in aweight ratio of 20/80 is poured into a polystyrene Petri dish and placedin a ventilation oven set at a temperature of 75° C. Once the solventhas completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 53

Preparation of a film containing HYAFF11p25 and polyurethane (PU),Cardiomat 610, Contron

0.670 ml of a 15% solution of PU in tetrahydrofuran:dioxane, 1:1, aredissolved in 8 ml of DMSO, while being shaken at a temperature of 70° C.for one hour. When the starting solvents have evaporated, the solutionis brought to a final volume of 10 ml with DMSO. A 1% solution of PU isthus obtained, which will be referred to as solution F.

Solution A is slowly added to solution F while being continuously shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture HYAFF11 p25/PU in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 54

Preparation of a film containing HYAFF11 p25 and polylactic acid (PLA)

100 mg of PLA are dissolved in DMSO while being shaken at a temperatureof 85° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PLA is thus obtained, which will be referred to assolution G.

Solution A is slowly added to solution G while being continuously shakenat a temperature of 85° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p25/PLA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 55

Preparation of a film of HYAFF11 p25 and polyphosphazene, for examplepoly (methoxyethoxy) phosphazene (PF3)

100 mg of PF3 are dissolved in DMSO while being shaken at a temperatureof 100° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PF3 is thus obtained, which will be referred to assolution H.

Solution A is slowly added to solution H while being constantly shakenat a temperature of 100° C. The resulting solution is shaken for onehour to allow complete amalgamation of the two components.

The resulting solution containing the mixture of HYAFF11 p25/PF3 in aweight ratio of 20/80 is poured into a polystyrene Petri dish and placedin a ventilation oven set at a temperature of 75° C. Once the solventhas completely evaporated, a transparent and homogeneous film isobtained.

The following example illustrates the preparation of a film by theevaporation of solvent from mixtures of hyaluronic acid esters withbenzyl alcohol (50% esterification, HYAFF11 p50) dissolved in DMSO andpolymers soluble in DMSO.

The solution of HYAFF11 p50 in DMSO is prepared as follows: 100 mg ofHYAFF11 p50 are dissolved in distilled water: DMSO, 1:1, while beingshaken at room temperature for 30 minutes. The water is then substitutedwith DMSO by adding the latter to the solution and heating to 90° C. toevaporate the water. Lastly, the solution is brought to a final volumeof 10 ml with DMSO. A 1% solution of HYAFF11 p50 in DMSO is thusobtained, and will be referred to as solution A in Example 56.

EXAMPLE 56

Preparation of a film containing HYAFF11 p50 and polyphosphazene, forexample poly (methoxyethoxy) phosphazene (PF3)

100 mg of PF3 are dissolved in DMSO while being shaken at a temperatureof 100° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PF3 is thus obtained, and this will be referred to assolution B.

Solution A is slowly added to solution B while being shaken at atemperature of 100° C. The solution is shaken for one hour to allowcomplete amalgamation of the two components.

The resulting solution containing the mixture of HYAFF11 p50/PF3 in aweight ratio of 20/80 is poured into a polystyrene Petri dish and placedin a ventilation oven set at a temperature of 75° C. Once the solventhas completely evaporated, a transparent and homogeneous film isobtained.

The following examples illustrate the preparation of films by theevaporation of solvent from mixtures of hyaluronic acid esters withbenzyl alcohol (75% esterification, HYAFF11 p75) dissolved in DMSO andpolymers soluble in DMSO.

100 mg of HYAFF11 p75 are dissolved in DMSO while being shaken at roomtemperature for 30 minutes. DMSO is then added to a final volume of 10ml. A 1% solution of HYAFF11 p75 is thus obtained, which will bereferred to as solution A in examples 57-62.

EXAMPLE 57

Preparation of a film containing HYAFF11 p75 and polyvinyl alcohol (PVA)

100 mg of PVA are dissolved in DMSO while being shaken at a temperatureof 100° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PVA is thus obtained, which will be referred to assolution B.

Solution A is slowly added to solution B while being constantly shakenat a temperature of 100° C. The resulting solution is shaken for onehour to allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p75/PVA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 58

Preparation of a film containing HYAFF11 p75 and Clarene L6, Solvay (C1L6), ethylene-vinyl copolymer with a low ethylene content (29 mole %)

100 mg of C1 L6 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 L6 is thus obtained, which will bereferred to as solution C.

Solution A is slowly added to solution C while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p75/C1 L6 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 59

Preparation of a film containing HYAFF11 p75 and Clarene P10, Solvay (C1P10), ethyl-vinyl alcohol copolymer with a medium ethylene content (36mole %)

100 mg of C1 P10 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 P10 is thus obtained which will bereferred to as solution D.

Solution A is slowly added to solution D while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, which contains HYAFF11 p75/C1 P10 in a weightratio of 20/80, is slowly poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 60

Preparation of a film containing HYAFF11 p75 and Clarene R20, Solvay (C1R20), ethylene-vinyl alcohol copolymer with a high ethylene content (40mole %)

100 mg of C1 R20 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 R20 is thus obtained, which will bereferred to as solution E.

Solution A is slowly added to solution E while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p75/C1 R20 ina weight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 61

Preparation of a film containing HYAFF11 p75 and polyurethane (PU),Cardiomat 610, Contron

0.670 ml of a 15% PU solution in tetrahydrofuran:dioxane, 1:1, aredissolved in 8 ml of DMSO while being shaken at a temperature of 70° C.for one hour. When the starting solvents have evaporated, the solutionis brought to a final volume of 10 ml with DMSO. A 1% solution of PU isthus obtained, which is referred to as solution F.

Solution A is slowly added to solution F while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p75/PU in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 62

Preparation of a film containing HYAFF11 p75 and polylactic acid (PLA)

100 mg of PLA are dissolved in DMSO while being constantly shaken at atemperature of 85° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of PLA is thus obtained, which will bereferred to as solution G.

Solution A is slowly added to solution G while being constantly shakenat a temperature of 85° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 p75/PLA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

The following examples describe the preparation of films by theevaporation of solvent from mixtures of hyaluronic acid esters withbenzyl alcohol (100% esterification, HYAFF11 dissolved in DMSO andpolymers soluble in DMSO. 100 mg of HYAFF11 are dissolved in DMSO whilebeing shaken at room temperature for 30 minutes. DMSO is then addeduntil a final volume of 10 ml is reached. A 15% solution of HYAFF11 inDMSO is thus obtained, which will be referred to as solution A inexamples 63-69.

EXAMPLE 63

Preparation of a film of HYAFF11 and polyvinyl alcohol (PVA) 100 mg ofPVA are dissolved in DMSO while being shaken at a temperature of 100° C.for one hour and are then brought to a final volume of 10 ml. A 1%solution of PVA is thus obtained, which will be referred to as solutionB.

Solution A is slowly added to solution B while being constantly shakenat a temperature of 100° C. The resulting solution is shaken for onehour to allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 /PVA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 64

Preparation of a film containing HYAFF11 and Clarene L6, Solvay (C1 L6),ethylene-vinyl alcohol copolymer with a low ethylene content (29 mole %)

100 mg of C1 L6 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought no a finalvolume of 10 ml. A 1% solution of C1 L6 is thus obtained, which will bereferred to as solution C.

Solution A is slowly added to solution C while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11/C1 L6 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 65

Preparation of a film containing HYAFF11 and Clarene P10, Solvay (C1P10), ethylene-vinyl alcohol copolymer with a medium ethylene content(36 mole %)

100 mg of C1 P10 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 P10 is thus obtained, which will bereferred to as solution D.

Solution A is slowly added to solution D while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 /C1 P10 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 66

Preparation of a film containing HYAFF11 and Clarene P20, Solvay (C1R20), ethylene-vinyl alcohol copolymer with a high ethylene content (40mole %)

100 mg of C1 R20 are dissolved in DMSO while being shaken at atemperature of 70° C. for one hour and are then brought to a finalvolume of 10 ml. A 1% solution of C1 R20 is thus obtained, which will bereferred to as solution E.

Solution A is slowly added to solution E while being constantly shakenat a temperature of 70° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 /C1 R20 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced into a ventilation oven set at a temperature of 75° C. Once thesolvent has evaporated, a transparent and homogeneous film is obtained.

EXAMPLE 67

Preparation of a film containing HYAFF11 and polyurethane (PU),Cardiomat 610, Controt

0.670 ml of a 15% solution of PU in tetrahydrofuran: dioxane, 1:1, aredissolved in DMSO while being shaken at a temperature of 70° C. for onehour. When the starting solvents have evaporated, the solution isbrought to a final volume of 10 ml with DMSO. A 1% solution of PU isthus obtained, which will be referred to as solution F.

Solution A is slowly added to solution F while being shaken at atemperature of 70° C. The resulting solution is shaken for one hour toallow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11 /PU in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 68

Preparation of a film containing HYAFF11 and polylactic acid (PLA)

100 mg of PLA are dissolved in DMSO while being shaken at a temperatureof 85° C. for one hour and are then brought to a final volume of 10 ml.A 1% solution of PLA is thus obtained, which will be referred to assolution G.

Solution A is slowly added to solution G while being constantly shakenat a temperature of 85° C. The resulting solution is shaken for one hourto allow complete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11/PLA in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

EXAMPLE 69

Preparation of a film containing HYAFF11and polyphosphazene, for examplepoly-phenoxy phosphazene

100 mg of PF4 are dissolved in DMSO while being shaken at a temperatureof 25° C. for one hour and brought to a final volume of 10 ml. A 1%solution of PF4 is thus obtained, which will be referred to as solutionH.

Solution A is slowly added to solution H while shaking at a temperatureof 25° C. The resulting solution is shaken for one hour to allowcomplete amalgamation of the two components.

The resulting solution, containing the mixture of HYAFF11/PF4 in aweight ratio of 20/80, is poured into a polystyrene Petri dish andplaced in a ventilation oven set at a temperature of 75° C. Once thesolvent has completely evaporated, a transparent and homogeneous film isobtained.

Crosslinking of preformed Polymers

In order to attain the objects of the present invention, all the polymerblends produced in the foregoing examples can be transformed intoadditional IPN or semi-IPN by the following methods.

EXAMPLE 70

Crosslinking of polymers using compounds capable of generating radicals##STR1## in which R can be H, halogen, SOCl₂, COCl, SO₃ H, COOH, orCOOR' can be crosslinked with many peroxides (Solovey et al., BellSystem Tech. J., 40:1400, 1961). The working temperatures are very high,leading to polymer degradation.

Aliphatic polyethers, such as polyethyleneoxide and polypropyleneoxidemay be crosslinked through peroxides (Y. Okada, J. Appl. Polym. Sci,7:695, 703, and 1153, 1963).

Polyamides, polysulphones, and polyesters can be crosslinked usingperoxides.

Besides peroxides, ethyltrichloroacetate, C₆ H₁₁ --CCl₃, R--S--S--R,etc., can be used to generate radicals.

With preformed polymers having C--C double bonds, "two reagent systems"can be used: one reagent generates radicals, while the second works asan intermolecular bridge between the double bonds. An example thereof isperoxide-dimaleimide.

Crosslinking of polymers having various functional groups

Carboxylic functional groups: polyacrylic acid, polymethacrylic acid andtheir copolymers with acrylate, methacrylate, styrene, and other vinylicmonomers, the copolymers of hydrolyzed maleic anhydride and sorbic acid,may be crosslinked with diamine, triamine, di- and polyisocyanate, di-,tri-, and polyols, N-methylol derivatives of formaldehyde, oxiraniccompounds, and carbodiimide. At high temperatures, the crosslink canoccur by the formation of intermolecular anidridic groups.

OH functional groups: polyvinylic alcohol and its copolymers withethylene, acrylate, methacrylate, beta-hydroxyethyl-acrylate, copolymerswith styrene-beta-hydroxyethylacrylate may be crosslinked throughformaldehyde, N-methylol derivatives of formaldehyde, dihalogenides ofcarboxylic acids, glyoxal glutaraldehyde, and other dialdehydes.

Mixtures of linear polymers having functional groups such as polyvinylicalcohol or its copolymers with polyacrylic or polymethacrylic acid maybe crosslinked through intermolecular esterification (Y. Tatara, J.Polym. Sci. Symp. 54:283, 1976). These mixtures may also be crosslinkedthrough the above-mentioned reagents for --COOH and --OH groups.

For cellulose, the following compounds have been used as crosslinkingreagents: urea-formaldehyde, dimethylol-urea, bis-dimethylol derivativesof cyclic ethylene-urea, and similar compounds such asmethylol-tetramethylene-urea, N-methylol derivatives of idanthoine, ofimizadolidone, of propylene-urea, and triazones. Other useful compoundsare glyoxal, glutaraldehyde, alpha-hydroxyadipaldehyde, and otherdialdehydes, diepoxides, such as cyclohexenedioxide, and others havingthe general structure: ##STR2## which can be derivatized withethyleneamine, sulphones, and alpha-chloroether.

Amino-ureic-urethanic functional groups: polyamides, polyurea, andpolyurethanes can be crosslinked using di- and polyisocyanates.

Besides the foregoing methods of obtaining IPN and semi-IPN bycrosslinking both of the components of the blend, semi-IPN can also beobtained by the polymerization of a monomer in the presence of acrosslinking agent and in the presence of the natural acidicpolysaccharide or a semisynthetic ester-type derivative thereof.

As an example, it has been possible to obtain a semi-IPN by polymerizingmetha-methacrylate, in which the polysaccharide has been dissolved, inthe presence of tetraethylene glycol dimethacrylate (TEGDM) as thecrosslinking agent and benzoin as initiator using a UVphotopolymerization process in bulk.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

We claim:
 1. A biomaterial, comprising a non-chemically crosslinkedinterpenetrating polymer network, IPN, having a first component and asecond component wherein said first component is a member selected fromthe group consisting of a hyaluronic acid ester and a salt of hyaluronicacid with an organic base, and said second component is a syntheticchemical polymer.
 2. The biomaterial of claim 1, wherein said firstcomponent is a hyaluronic acid ester and wherein said hyaluronic acidester is a 100% hyaluronic acid ester or a partial hyaluronic acidester.
 3. The biomaterial of claim 2, wherein said hyaluronic acid esteris 100% hyaluronic acid ester and wherein said 100% hyaluronic acidester is a member selected from the group consisting of a 100% benzylester of hyaluronic acid and a 100% ethyl ester of hyaluronic acid, andsaid partial hyaluronic acid ester is a member selected from the groupconsisting of a 10% partial benzyl ester of hyaluronic acid, a 25%partial benzyl ester of hyaluronic acid, a 50% partial benzyl ester ofhyaluronic acid, and a 75% partial benzyl ester of hyaluronic acid. 4.The biomaterial of claim 1, wherein said first component is a hyaluronicacid ester and wherein said hyaluronic acid ester is a partial or totalhyaluronic acid ester containing an alcohol having a chain length of 14carbon atoms or less.
 5. The biomaterial of claim 1, wherein said firstcomponent is a hyaluronic acid ester and wherein said hyaluronic acidester is an ester of hyaluronic acid with an aliphatic, araliphatic,cycloaliphatic, or heterocyclic alcohol.
 6. The biomaterial of claim 1,wherein said organic base is a pharmacologically active molecule.
 7. Thebiomaterial of claim 6, wherein said pharmacologically active moleculeis a member selected from the group consisting of an antiinfectiveagent, an antibiotic agent, an antimicrobial agent, an antiinflammatoryagent, a cytostatic agent, a cytotoxic agent, an antiviral agent, ananaesthetic agent, an antiseptic agent, and a disinfecting agent.
 8. Thebiomaterial of claim 1, wherein said hyaluronic acid ester is an esterwith a pharmacologically active molecule.
 9. The biomaterial of claim 8,wherein said pharmacologically active molecule is a member selected fromthe group consisting of an antiinfective agent, an antibiotic agent, anantimicrobial agent, an antiinflammatory agent, a cytostatic agent, acytotoxic agent, an antiviral agent, an anaesthetic agent, an antisepticagent, and a disinfecting agent.
 10. The biomaterial of any one ofclaims 1-9, wherein the polymers comprising said IPN are soluble indimethylsulfoxide.
 11. The biomaterial of claim 1, wherein saidsynthetic chemical polymer is grafted onto said hyaluronic acid ester orsalt of hyaluronic acid with an organic base.
 12. The biomaterial ofclaim 11, wherein the grafting is achieved via functional groups on saidhyaluronic acid ester or salt of hyaluronic acid with an organic baseand said synthetic chemical polymer.
 13. The biomaterial of claim 12,wherein said IPN is formed prior to grafting.
 14. The biomaterial ofclaim 1, wherein said biomaterial is in a form selected from the groupconsisting of a film, a membrane, a sponge, a hydrogel, a guide channel,a thread, a gauze, and a non-woven tissue.
 15. The biomaterial of claim1, further comprising a therapeutic agent selected from the groupconsisting of an antiinfective agent, an antibiotic agent, anantimicrobial agent, an antiinflammatory agent, a cytostatic agent, acytotoxic agent, an antiviral agent, an anaesthetic agent, an antisepticagent, and a disinfecting agent.
 16. The biomaterial of claim 1, whereinsaid synthetic chemical polymer is a member selected from the groupconsisting of polyacrylic acid, polyvinylpyrrolidone, potyacrylamide,polyethylene oxide, vinyl alcohol-vinyl acetate copolymer, polyvinylalcohol, poly-(trifluoroethoxy)phosphazene, poly-(di(p-sodiosulfoxyphenoxy) phosphazene), poly(methoxyethoxy) phosphazene,poly(phenoxy) phosphazene, ethylene-vinyl alcohol copolymerpolyurethane, and polylactic acid.
 17. The biomaterial of claim 2,wherein in said partial hyalurontc acid ester, the free carboxylic acidgroups are salified with a metal or an organic base.
 18. The biomaterialof claim 17, wherein said metal is an alkali metal or alkaline earthmetal.
 19. The biomaterial of claim 17, wherein said organic base isammonia or a nitrogenous organic base.